Disrupted brain thyroid hormone homeostasis and altered thyroid hormone-dependent brain gene expression in autism spectrum disorders.

The present study examined human postmortem brains for changes consistent with the hypothesis of local brain TH deficiency in autism spectrum disorders (ASD). Brain levels of oxidative stress marker - 3-nitrotyrosine (3-NT), iodothyronine deiodinase type 2(D2) and type 3 (D3), 3',3,5-triiodothyronine (T3) content, mercury content and gene expression levels were analyzed and compared in the several regions of postmortem brains derived from both male and female control and ASD cases, age 4-16 years. We report that some parameters measured, such as D2 are subject to rapid postmortem inactivation, while others that were analyzed showed both brain region- and sex-dependent changes. Levels of 3-NT were overall increased, T3 was decreased in the cortical regions of ASD brains, while mercury levels measured only in the extracortical regions were not different. The expression of several thyroid hormone (TH)-dependent genes was altered in ASD. Data reported here suggest the possibility of brain region-specific disruption of TH homeostasis and gene expression in autism.

Effects of perinatal lipopolysaccharide (LPS) exposure on the developing rat brain; modeling the effect of maternal infection on the developing human CNS.

In the present study, we examined the effect of perinatal Escherichia coli lipopolysaccharide (LPS) exposure on the developing rat cerebellum and tested the hypothesis that maternal infections impact brain structure and function by mechanisms involving increase in oxidative stress and changes in brain type 2 iodothyronine deiodinase (D2)- and thyroid hormone (TH)-responsive genes. Spontaneously hypertensive rat (SHR) and Sprague-Dawley (SD) rat dams were challenged with LPS (200 µg/kg body weight) exposure during pregnancy (G10-G15) and lactation (P5-P10), the time periods corresponding, respectively, to the first/second and the third trimesters of human pregnancy. LPS exposure resulted in a significantly decreased motor learning in SD male (29.8 %) and in female (55.0 %) pups (p < 0.05); changes in rollover and startle response showed only a trend. The LPS challenge also resulted in a trend (p = 0.09) toward increased cerebellar levels of the oxidative stress marker 3-nitrotyrosine (3-NT) in SD male (16.2 %) and female (21.2 %) neonates, while 3-NT levels were significantly decreased (p < 0.05) in SHR female pups. D2 activity, responsible for local intra-brain conversion of thyroxine (T4) to the active hormone, 3',3,5-triiodothyronine (T3), was significantly (p < 0.05) decreased in LPS-challenged SHR male (40.3 %) and SD female (47.4 %) pups. Several genes were affected by LPS. Notably, D2 (DIO2) and brain-derived neurotrophic factor (BDNF) were significantly elevated in SHR females, while transthyretin (TTR) expression was decreased in both SD males and females (P < 0.05). In vitro chronic exposure of cerebellar cultures to LPS resulted in decreased arborization of Purkinje cells while D2 was only increased transiently. Our data demonstrate that perinatal LPS exposure impacts the developing cerebellum in strain- and sex-dependent manner via complex mechanisms that involve changes in oxidative stress, enzymes involved in maintaining local TH homeostasis, and downstream gene expression.

Sex-dependent changes in cerebellar thyroid hormone-dependent gene expression following perinatal exposure to thimerosal in rats.

Mammalian brain development is regulated by the action of thyroid hormone (TH) on target genes. We have previously shown that the perinatal exposure to thimerosal (TM, metabolized to ethylmercury) exerts neurotoxic effects on the developing cerebellum and is associated with a decrease in cerebellar D2 activity, which could result in local brain T3 deficiency. We have also begun to examine TM effect on gene expression. The objective of this study was to expand on our initial observation of altered cerebellar gene expression following perinatal TM exposure and to examine additional genes that include both TH-dependent as well as other genes critical for cerebellar development in male and female neonates exposed perinatally (G10-G15 and P5 to P10) to TM. We report here for the first time that expression of suppressor-of-white-apricot-1 (SWAP-1), a gene negatively regulated by T3, was increased in TM-exposed males (61.1% increase), but not in females; (p<0.05). Positively regulated T3-target genes, Purkinje cell protein 2 (Pcp2; p=0.07) and Forkhead box protein P4 (FoxP4; p=0.08), showed a trend towards decreased expression in TM-exposed males. The expression of deiodinase 2 (DIO2) showed a trend towards an increase in TM-exposed females, while deiodinase 3 (DIO3), transthyretin (TTR), brain derived neurotrophic factor (BDNF) and reelin (RELN) was not significantly altered in either sex. Since regulation of gene splicing is vital to neuronal proliferation and differentiation, altered expression of SWAP-1 may exert wide ranging effects on multiple genes involved in the regulation of cerebellar development. We have previously identified activation of another TH-dependent gene, outer dense fiber of sperm tails 4, in the TM exposed male pups. Together, these results also show sex-dependent differences between the toxic impacts of TM in males and females. Interestingly, the genes that were activated by TM are negatively regulated by TH, supporting our hypothesis of local brain hypothyroidism being induced by TM and suggesting a novel mechanism of action TM in the developing brain.

Maternal thimerosal exposure results in aberrant cerebellar oxidative stress, thyroid hormone metabolism, and motor behavior in rat pups; sex- and strain-dependent effects.

Methylmercury (Met-Hg) and ethylmercury (Et-Hg) are powerful toxicants with a range of harmful neurological effects in humans and animals. While Met-Hg is a recognized trigger of oxidative stress and an endocrine disruptor impacting neurodevelopment, the developmental neurotoxicity of Et-Hg, a metabolite of thimerosal (TM), has not been explored. We hypothesized that TM exposure during the perinatal period impairs central nervous system development, and specifically the cerebellum, by the mechanism involving oxidative stress. To test this, spontaneously hypertensive rats (SHR) or Sprague-Dawley (SD) rat dams were exposed to TM (200 µg/kg body weight) during pregnancy (G10-G15) and lactation (P5-P10). Male and female neonates were evaluated for auditory and motor function; cerebella were analyzed for oxidative stress and thyroid metabolism. TM exposure resulted in a delayed startle response in SD neonates and decreased motor learning in SHR male (22.6%), in SD male (29.8%), and in SD female (55.0%) neonates. TM exposure also resulted in a significant increase in cerebellar levels of the oxidative stress marker 3-nitrotyrosine in SHR female (35.1%) and SD male (14.0%) neonates. The activity of cerebellar type 2 deiodinase, responsible for local intra-brain conversion of thyroxine to the active hormone, 3',3,5-triiodothyronine (T3), was significantly decreased in TM-exposed SHR male (60.9%) pups. This coincided with an increased (47.0%) expression of a gene negatively regulated by T3, Odf4 suggesting local intracerebellar T3 deficiency. Our data thus demonstrate a negative neurodevelopmental impact of perinatal TM exposure which appears to be both strain- and sex-dependent.

Type-2 iodothyronine 5'deiodinase (D2) in skeletal muscle of C57Bl/6 mice. II. Evidence for a role of D2 in the hypermetabolism of thyroid hormone receptor alpha-deficient mice.

Mice with ablation of the Thra gene have cold intolerance due to an as yet undefined defect in the activation of brown adipose tissue (BAT) uncoupling protein (UCP). They develop an alternate form of facultative thermogenesis, activated at temperatures below thermoneutrality and associated with hypermetabolism and reduced sensitivity to diet-induced obesity. A consistent finding in Thra-0/0 mice is increased type-2 iodothyronine deiodinase (D2) mRNA in skeletal muscle and other tissues. With an improved assay to measure D2 activity, we show here that this enzyme activity is increased in proportion to the mRNA and as a function of the ambient cold. The activation is mediated by the sympathetic nervous system in Thra-0/0, as it is in wild-type genotype mice, but the sympathetic nervous system effect is greater in Thra-0/0 mice. Using D2-ablated mice (Dio2-/-), we reported elsewhere and show here that, in spite of sharing a severe deficiency in BAT thermogenesis with Thra-0/0 and UCP1-knockout mice, they do not have an increase in oxygen consumption, and they gain more weight than wild-type controls when fed a high-fat diet. UCP3 mRNA is highly responsive to thyroid hormone, and it is increased in Thra-0/0 mice, particularly when fed high-fat diets. We show here that muscle UCP3 mRNA in hypothyroid Thra-0/0 mice is responsive to small dose-short regimens of T(4), indicating a role for locally, D2-generated T(3). Lastly, we show that bile acids stimulate not only BAT but also muscle D2 activity, and this is associated with stimulation of muscle UCP3 mRNA expression provided T(4) is present. These observations strongly support the concept that enhanced D2 activity in Thra-0/0 plays a critical role in their alternate form of facultative thermogenesis, stimulating increased fat oxidation by increasing local T(3) generation in skeletal muscle.

Physiological role and regulation of iodothyronine deiodinases: a 2011 update.

T4 is a prohormone secreted by the thyroid. T4 has a long half life in circulation and it is tightly regulated to remain constant in a variety of circumstances. However, the availability of iodothyronine selenodeiodinases allow both the initiation or the cessation of thyroid hormone action and can result in surprisingly acute changes in the intracellular concentration of the active hormone T3, in a tissue- specific and chronologically-determined fashion, in spite of the constant circulating levels of the prohormone. This fine-tuning of thyroid hormone signaling is becoming widely appreciated in the context of situations where the rapid modifications in intracellular T3 concentrations are necessary for developmental changes or tissue repair. Given the increasing availability of genetic models of deiodinase deficiency, new insights into the role of these important enzymes are being recognized. In this review, we have incorporated new information regarding the special role played by these enzymes into our current knowledge of thyroid physiology, emphasizing the clinical significance of these new insights.

Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor.

The farnesoid X-activated receptor (FXR; NR1H4) is a member of the nuclear hormone receptor superfamily and functions as a heterodimer with the 9-cis-retinoic acid receptor (RXR). In order to determine the optimal DNA binding sequence for the FXR/RXR heterodimer, we have utilized the selected and amplified binding sequence imprinting technique. This technique identified a number of related sequences that interacted with FXR/RXR in vitro. The consensus sequence contained an inverted repeat of the sequence AGGTCA with a 1-base pair spacing (IR-1). This sequence was shown to be a high affinity binding site for FXR/RXR in vitro and to confer ligand-dependent transcriptional activation by FXR/RXR to a heterologous promoter. Electrophoretic mobility shift assays and transient transfection assays were used to investigate the importance of the core half-site sequences, spacing nucleotide, flanking sequences, and orientation and spacing of the core half-sites on DNA binding and ligand-dependent transcriptional activation by FXR/RXR. These studies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, although FXR/RXR can bind to and activate through a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, FXR/RXR can bind to and transactivate through direct repeats. Three genes were identified that contain IR-1 sequences in their proximal promoters. These elements were shown to bind FXR/RXR in vitro and to confer FXR/RXR-dependent transcriptional activation to a heterologous promoter in response to a bile acid or synthetic retinoid. The endogenous mRNA levels of one of these genes, phospholipid transfer protein, were shown to be induced by FXR and FXR ligands. The identification of the IR-1 and related elements as high affinity binding sites and functional response elements for FXR/RXR and the identification of a target gene for FXR/RXR should assist in the identification of additional genes regulated by FXR/RXR.

Bile acids: natural ligands for an orphan nuclear receptor.

Bile acids regulate the transcription of genes that control cholesterol homeostasis through molecular mechanisms that are poorly understood. Physiological concentrations of free and conjugated chenodeoxycholic acid, lithocholic acid, and deoxycholic acid activated the farnesoid X receptor (FXR; NR1H4), an orphan nuclear receptor. As ligands, these bile acids and their conjugates modulated interaction of FXR with a peptide derived from steroid receptor coactivator 1. These results provide evidence for a nuclear bile acid signaling pathway that may regulate cholesterol homeostasis.

Activation of the orphan receptor RIP14 by retinoids.

Retinoids are crucial regulators of a wide variety of processes in both developing and adult animals. These effects are thought to be mediated by the retinoic acid (RA) receptors and the retinoid X receptors (RXRs). We have identified an additional retinoid-activated receptor that is neither a retinoic acid receptors nor an RXR. RXR-interacting protein 14 (RIP14), a recently described orphan member of the nuclear receptor superfamily, can be activated by either all-trans-RA (tRA) or the synthetic retinoid TTNPB [[E]-4-[2-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl)propen-1-yl]benzoic acid].RIP14 binds to DNA as a heterodimer with RXR. In the presence of either tRA or TTNPB, the addition of 9-cis-RA or the RXR-specific agonist LG1069 [4-[1-(3, 5, 5, 8, 8-pentamethyl-5, 6, 7, 8-tertrahydro-2-naphthyl)ethenyl]benzoic acid] results in additional activation. Mutations of the ligand-dependent transcriptional activation functions indicate that TTNPB activates the RIP14 component of the RIP14-RXR heterodimer, that 9-cis-RA and LG1069 activate RXR, and that tRA activates via both RIP14 and RXR. Despite the very effective activation of RIP14 by tRA or TTNPB, relatively high concentrations of these compounds are required, and no evidence for direct binding of either compound was obtained using several approaches. These results suggest that RIP14 is the receptor for an as-yet-unidentified retinoid metabolite.

Structural features of thyroid hormone response elements that increase susceptibility to inhibition by an RTH mutant thyroid hormone receptor.

The chicken lysozyme silencer F2 (F2) thyroid hormone response element (TRE) contains an unusual everted palindromic arrangement, has a high affinity for thyroid hormone receptor (TR) homodimers, and is especially sensitive to dominant negative inhibition by, the T3 resistance (RTH) mutant TR beta P453H. We used various TREs and TR mutations to determine the mechanisms for this sensitivity. Changing the F2 orientation from an everted palindrome to a direct repeat with a 4-bp gap (DR+4) (F2-DR) decreased the sensitivity to inhibition at high T3 concentrations, while a loss of this sensitivity occurred with a palindromic arrangement of these same half-sites. F2 contains the dinucleotide TG 5' to each consensus half-site conforming to the optimal TR-binding octamer, YRRGGTCA. A T to A change in position 1 of both F2 half-sites markedly reduced T3-induction, yet only slightly reduced TR homodimer or TR-retinoid X receptor (RXR) heterodimer binding. The TR beta ninth heptad mutation, L428R, prevents TR heterodimerization with RXR and eliminates the inhibitory effect of the P453H mutant TR on the F2-DR, but not the F2 element. Structural features of a TRE that favor strong TR binding of both TR homodimers and TR-RXR heterodimers containing the mutant TR, such as the everted palindromic conformation or the optimal TR-binding consensus octamer, enhance the sensitivity of a TRE to inhibition by the mutant TR. Thus, both half-site orientation and sequence contribute to the sensitivity of a given TRE to dominant negative inhibition by a mutant TR.

Enhancement of thyroid hormone receptor isoform specificity by insertion of a distant half-site into a thyroid hormone response element.

The two isoforms of thyroid hormone receptor (TR), alpha and beta, are highly homologous, except in the amino-terminal domain. Specific physiological roles for the receptor isoforms have not yet been defined. In transient transfection assays, TRalpha is twice as potent a thyroid hormone (T3)-dependent transactivator as TRbeta on a number of thyroid hormone response elements (TREs). Using chimeras of TRalpha and -beta, we have determined that the higher transactivation by TRalpha requires the entire ligand-binding domain. The amino-terminal and DNA-binding domains of the two isoforms are interchangeable. These studies were facilitated by the use of a synthetic TRE composed of a direct repeat separated by 4 bp which also included a third half-site 19 bp 3' to this on the opposite strand. In the presence of T3, this TRE confers a 5-fold higher response to TRalpha than to TRbeta, but there is no difference in expression without T3. Functional studies indicate that all three half-sites are needed for the increased responsiveness to TRalpha, but gel shift analyses show no striking differences in the ratios of TRalpha to TRbeta binding compared to other wild-type TREs. These results suggest that important functional differences are present in the ligand-binding domain of TRalpha and -beta despite their high homology.

A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5'-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3'-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp -100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the -2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) beta or JEG nuclear extracts (containing RXR alpha) and bacterially expressed chicken T3 receptor alpha 1 (TR alpha) can occupy both half-sites although the 3' half-site is dominant. T3 causes dissociation of TR alpha from the 5' half-site but increases binding to the 3' half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR+15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3' half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp -700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.

Retinoid-X receptor (RXR) differentially augments thyroid hormone response in cell lines as a function of the response element and endogenous RXR content.

Retinoid-X receptor (RXR) forms heterodimers with thyroid hormone receptor (TR) and significantly enhances binding to thyroid hormone response elements (TREs). Expression of RXR in a transient transfection assay augments the T3 response, but the influences of the specific cell line and TRE used have not been systematically studied. We determined RXR alpha and -beta augmentation of the TR alpha-mediated T3 response in transient transfection assays of COS, JEG, and mouse embryonic stem (ES) cell lines for a series of eight wild-type thyroid hormone (T3) and retinoic acid response elements (previously shown to bind TR). RXR augmented T3-induced expression in COS and ES cells (1.5- to 4-fold greater expression with added RXR compared to TR alone), but had minimal effect on augmentation of response in JEG cells. For most elements studied there was a proportional augmentation of basal and T3-stimulated expression. TREs from rat GH and laminin-B1, however, had relatively higher levels of T3-induced expression as a result of RXR cotransfection (T3 induction ratios increased 2-fold or greater). Previous characterization of these elements demonstrates that they contain more than two hexameric binding domains, all of which can simultaneously bind TR. The influence of endogenous RXR expression in a cell line on RXR augmentation of the T3 response was determined. RXR alpha and -beta messenger RNA (mRNA) expression was quantitated by Northern blot in each cell line. COS and JEG cells expressed almost exclusively RXR alpha mRNA, although expression was almost 2-fold higher in JEG compared to COS cells (12 +/- 2.5 vs. 6.8 +/- 0.5 density units relative to actin; mean +/- SE; P < 0.05). ES cells expressed only RXR beta mRNA, but at a very low level (0.4 +/- 0.1). Nuclear extracts prepared from JEG and COS cells augmented TR binding proportional to the endogenous RXR mRNA expression, and the heterodimer band was supershifted by the addition of antibody to RXR alpha. Nuclear extracts from ES cells had no detectable TR heterodimer binding to a range of response elements. RXR augmentation of the T3 response differs among cell lines and is greater in those with reduced endogenous RXR. Furthermore, the functional augmentation of the T3 response ratio by RXR is likely to require additional sequences contained in only a subset of elements in which RXR augments TR binding.

Thyroid hormone receptor binds with unique properties to response elements that contain hexamer domains in an inverted palindrome arrangement.

Thyroid hormone receptor (T3R)-accessory protein heterodimers preferentially bind to thyroid hormone response elements (TREs), which contain hexamer domains arranged as a direct repeat separated by a 4-basepair gap (DR + 4). T3R homodimers, however, preferentially bind to elements that consist of an inverted palindrome. We now report on unique T3R binding patterns and functional characteristics of two such elements that mediate T3 regulation. We performed mutational analysis of the chicken lysozyme silencer F2 (LysF2) TRE and demonstrated that the two functional binding domains are arranged as an inverted palindrome separated by 6 basepairs. Both the LysF2 TRE and the similarly arranged myelin basic protein TRE bind T3R dimers at very low T3R concentrations. Despite the high relative affinity for T3R dimer binding, the T3 induction conferred by these elements is low compared to that of previously characterized TREs with a DR + 4 arrangement. The laminin-B1 gene element, previously shown to bind retinoic acid receptor, contains at least four hexameric binding domains. All of the domains can bind T3R simultaneously and are involved in conferring T3 induction, but bind with a different pattern than that reported for retinoic acid receptor. T3R homodimer binding to a series of mutant laminin-B1 elements and T3 induction were significantly correlated (r = 0.82; P < 0.05). T3R homodimer binding to LysF2 element mutants was not correlated with T3 induction (r = 0.32; P > 0.05); however, T3R-nuclear protein heterodimer binding was significantly correlated (r = 0.67; P < 0.05). T3R-nuclear protein heterodimers, but not homodimers, bound consistently to mutations of the LysF2 element that altered the gap between hexamers. The overall discordance between strong T3R binding to these elements and weak T3 induction indicates that the unusual hexamer arrangement places the T3R complex in an unfavorable configuration for maximal T3-dependent transactivation. The differential T3 sensitivity of generalized resistance to thyroid hormone-associated T3R mutants to the LysF2 element compared with the DR + 4 arrangement suggests that these unique features may have physiological significance.

Dominant negative inhibition by mutant thyroid hormone receptors is thyroid hormone response element and receptor isoform specific.

The heterogeneity of tissue-specific manifestations of generalized resistance to thyroid hormone (GRTH) could result from differential interactions between the mutant thyroid hormone (T3) receptor-beta (TR beta) on T3 response elements (TREs) in different T3-responsive genes. To explore this hypothesis, the mutant TR beta associated with kindred A, P448H; a TR beta mutant, P448L; and a comparable TR alpha mutant (P398H) were tested for intrinsic function and for inhibition of wild-type TR alpha- and -beta-induced expression from four structurally distinct TREs, the rGH ABC*, the rGH palindrome (PAL), the rat malic enzyme (ME), and the chicken lysozyme silencer F2 (F2). The relative function of the mutants was similarly reduced on the four TREs studied and was T3 concentration dependent. The TR alpha mutant retained the intrinsically greater potency characteristic of this isoform, but remained impaired with respect to wild-type TR alpha even at 500 nM T3. In general, dominant negative inhibition of wild-type TR alpha and -beta function was dependent upon the T3 concentration, as expected from the decreased affinity for ligand conferred by this mutation. A T3 concentration sufficient to relieve the inhibition of wild-type TR function on the ABC*, PAL, and ME TREs (50 nM) had no effect on inhibition of the F2 TRE by the mutant TRs. Receptor isoform preferential inhibition was observed on the ABC*, PAL, and ME TREs by the mutant TRs. Thus, both TRE structure and the isoform of endogenously active receptor could determine the degree of inhibition of a specific gene in GRTH individuals. Further, the lack of dominant negative potentials does not explain the absence of TR alpha mutations in GRTH kindreds.

Protein discharge from immature secretory granules displays both regulated and constitutive characteristics.

At physiological glucose concentrations, isolated pancreatic islets release a minor portion of their newly synthesized insulin and precursors in a phase of secretion which is largely complete by 4 h of chase. Discharge during this period can be amplified by secretagogues, yet is not abolished by conditions which fully suppress regulated release from dense core secretory granules. The ability to stimulate the secretion and the biochemical profile of released proinsulin-related peptides indicate that secretion during this period originates from immature granules. The stoichiometry of release of labeled C-peptide:insulin during this phase is 1:1 at high glucose concentrations. However, at physiologic or low concentrations, C-peptide is released in molar excess of insulin as if the exocytotic vesicles carrying this secretion were budding from a post-Golgi compartment in which the lumen was composed of condensing insulin and soluble C-peptide. These findings can be explained by a model for regulated secretory protein traffic in which direct exocytosis of young granules is stimulated by higher glucose concentrations and vesicle budding from immature granules occurs at lower concentrations. Thus, insulin targeting from immature granules exhibits both regulated and constitutive-like characteristics.

Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium.

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)