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K A Strait

Publications and source records attributed to K A Strait.

18 recordsLinked to original sources
PubMed1996

Thyroid hormone receptor isoform content in cultured type 1 and type 2 astrocytes.

Immunohistochemical studies previously reported from this laboratory showed that astrocytes in adult rat brain appear devoid of all thyroid hormone receptor (TR) isoforms. These findings, however, contrast with reports of measurable nuclear T3 binding in astrocytes in cell culture. To address this discrepancy, TR protein and messenger RNA (mRNA) content of type 1 and type 2 astrocytes in culture were assayed. Type 1 cells represent astrocytes present in brain in vivo. Type 2 astrocytes differentiate in culture from bipotential progenitor O-2A cells in the presence of serum. Under serum-free conditions, these progenitor cells differentiate into oligodendroglia. Total nuclear T3 binding capacity in both type 1 and type 2 astrocytes was approximately 3000 sites/cell. Northern blots showed the presence of mRNA for TRbeta1, TRalpha1, and TRalpha2 in type 2 cells but failed to reveal the presence of these mRNAs in type 1 astrocytes. Moreover, Northern blots also failed to reveal TRbeta2 mRNA in both type 1 and type 2 astrocytes. These findings, therefore, raised a question as to which receptor isoform was responsible for the nuclear binding capacity observed in type 1 astrocytes. As anticipated, immunocytochemical analysis demonstrated prominent nuclear signals for TRbeta1, TRalpha1, and TRalpha2 mRNA in type 2 astrocytes but failed to demonstrate TRbeta1, TRalpha1, or TRalpha2 in type 2 astrocytes. Application of RT-PCR, however, revealed the presence of low levels of TRbeta2 mRNA in type 1 astrocytes. When stained with a specific anti-TRbeta2 antiserum, both type 1 and type 2 astrocytes showed a strong fluorescent signal concentrated in the nucleus. These data indicate that under the special conditions of cell culture, expression of the TRbeta2 isoform in type 1 accounts for the measured nuclear T3 binding capacity.

Animals↗
PubMed1995

Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation.

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freund's adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Identification of thyroid hormone response elements in rodent Pcp-2, a developmentally regulated gene of cerebellar Purkinje cells.

In a previous study, we have shown that in vivo expression of the cerebellar Purkinje cell-specific gene Pcp-2 is regulated by thyroid hormone (T3) during neonatal development. In addition, transient cotransfection studies using thyroid hormone receptors (TRs) and a Pcp-2-lacZ construct pointed to direct regulation of Pcp-2 gene expression by T3. Therefore, we have initiated the following series of studies to define more precisely the location of the thyroid hormone regulatory elements in the Pcp-2 gene. By transfection and in vitro receptor binding analyses, we have identified two thyroid hormone response elements, A1 (-295/-268) and B1 (+207/+227). A1 contains a central half-site flanked by two similar half-sites. B1 contains two pairs of alternate half-sites. When these elements were ligated to the modified mouse mammary tumor virus promoter (delta MMTV), both induced a 8-14-fold expression of the reporter gene, but only in the presence of T3. Gel mobility assays demonstrated that both A1 and B1 bind TRs in the presence of thyroid hormone receptor auxiliary proteins or the retinoid X beta receptor. Mutations of the G residues to T within the individual half-site sequences of A1 caused a variable decrease in the transactivation of the MMTV-CAT construct and a corresponding reduction in TR binding in vitro. Thus, mutational analysis of A1 pointed to the interaction of the flanking half-site motifs with the central AGGTCA half-site. Interestingly, lengthening of the A1 sequence at its 3'-end caused a progressive dampening of the T3 response. The results suggest that the neighboring sequence may function as a silencer of the A1 element. Since thyroid hormone regulation of Pcp-2 is manifest only during the first 2 weeks after birth, we hypothesize that A1 and B1 act as T3-dependent response elements operative only during early neonatal Purkinje cell development and that their function is suppressed by a neighboring silencer element operative when expression of Pcp-2 becomes hormone-independent.

Animals↗
PubMed1994

Immunofluorescent localization of thyroid hormone receptor isoforms in glial cells of rat brain.

The three currently recognized T3 binding thyroid hormone receptor (TR) isoforms, TR alpha 1, TR beta 1, and TR beta 2, arise from two distinct genes (alpha and beta), whereas two closely related non-T3-binding receptor variants, collectively designated TR alpha 2, arise from alternate splicing of the alpha gene transcript. Using a panel of specific antisera to these isoforms we have assessed the presence or absence of TRs in oligodendrocytes and astrocytes of rat cerebrum and cerebellum. Inferences as to colocalization of the receptor isoforms and cell-specific marker proteins were based on immunohistochemical analysis of the differential emissions of paired immunofluorescent probes. Antisera against myelin basic protein (MBP) identified oligodendroglia, and glial fibrillary acidic protein identified astrocytes. MBP-positive oligodendrocytes displayed positive fluorescent signals with each of the three TR isoform-specific antisera and the antiserum to the receptor variants. These findings are consistent with the concept that the MBP gene is a direct target for thyroid hormone action. TR immunoreactivity appeared to localize primarily to the nuclei of these cells. In contrast, we observed no immunofluorescent signals for any of the TR isoforms in glial fibrillary acidic protein-positive astrocytes. These findings raise the possibility that any effect of thyroid hormone on astrocyte function and structure is mediated indirectly as a result of interaction of thyroid hormone with receptors situated in nonastrocyte cells or as a result of nonnuclear mechanisms.

Animals↗
PubMed1993

Immunocytochemical delineation of thyroid hormone receptor beta 2-like immunoreactivity in the rat central nervous system.

The thyroid hormone receptors (TR) are nuclear proteins that include TR alpha and TR beta subtypes, each encoded by a separate gene. Both TR alpha and TR beta give rise to several isoforms of which three, TR alpha 1, TR beta 1, and TR beta 2 bind T3 and mediate the action of thyroid hormone. Although TR beta 2 was initially thought to be confined to the anterior pituitary, we recently observed small quantities of TR beta 2 messenger RNA (mRNA) by polymerase chain reaction analysis of discrete hypothalamic regions. To further examine the distribution of TR beta 2 in the brain, we performed immunocytochemical studies using a highly specific antiserum to TR beta 2, raised against a unique amino acid sequence (TR beta 2[131-145]) that is not present in the other known TRs. This antiserum immunoprecipitated TR beta 2 but not TR alpha 1 or TR beta 1. Immunoreactive TR beta 2 was widely distributed throughout the brain and primarily localized to the cell nucleus. Particularly intense immunostaining was present in the cerebral cortex, cerebellum, and hypothalamus, including regions where TR beta 2 mRNA had not previously been identified. In addition, immunoprecipitation of nuclear extracts with anti-TR beta 2 reduced total T3 binding capacity by approximately 20%, suggesting that immunoreactive TR beta 2 comprises a substantial portion of the total content of nuclear thyroid hormone binding proteins. These studies demonstrate that immunoreactive TR beta 2 is more widely represented in the central nervous system than previously suspected and may play an important role in mediating the action of T3 in many different regions of the brain. The finding of TR beta 2-like material could be due to a disproportionately high ratio of the TR beta 2 translation product and its mRNA in certain regions of the brain, or could indicate the existence of a novel TR beta 2-related protein that is important for T3 binding.

Amino Acid Sequence↗
PubMed1993

Molecular mechanisms of thyroid hormone action. A physiologic perspective.

At present, it appears abundantly clear that thyroid hormone exerts its major action at the nuclear level by regulating the level of mRNAs of specific genes. There are at least three TR isoforms that mediate hormonal effects at the tissue level. Characterization of the functional domains of these receptor isoforms is as yet incomplete, and the possibility that these receptors could have ligand-independent functions is a matter under current investigation. TRs are now recognized as members of a large superfamily of transactivating proteins involved in the regulation of gene expression. Recent studies have shown an unexpected degree of complexity in the nature of the association of the T3 receptors and the DNA of target genes. They have vividly pointed out the multiple interactions possible between the T3-receptor complex and other proteins participating in the process of gene regulation. These insights have provided a solid base for understanding differences in the gradation of thyroid hormone effect from one tissue to another. The microdissection of the molecular process that has occurred in the past 20 years has proceeded in part through the application of relatively artificial in vitro systems and assays. Whereas such approaches have undoubtedly reaped rich rewards in pointing out potential or possible mechanisms, they do not define the actual workings in the animal. Additional studies designed to examine at the molecular level the operation in vivo of physiologic networks influenced by thyroid hormones appear as an essential next step in understanding the biology of the hormone system. The application of transgenic models should materially assist such efforts.

Animals↗
PubMed1992

Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.

Amino Acid Sequence↗
PubMed1992

Effect of thyroid hormone status on the expression of the mRNAs of components of the lipolytic regulatory cascade in brown adipose tissue.

1. The levels of mRNAs for RII beta and G beta were about 50% lower in brown adipose tissue (BAT) from hyperthyroid than from hypothyroid rats. 2. Treatment of hypothyroid rats with T3 resulted in a 50% decrease in mRNAs for RII beta and G beta in BAT occurring by 12 hr after treatment. 3. The levels of mRNAs for hormone-sensitive lipase, G alpha s and C alpha in BAT were unchanged by thyroid hormone status. 4. The results suggest that thyroid hormone may be involved in negative regulation of the expression of RII beta and G beta at the transcriptional level in BAT.

Adipose Tissue, Brown↗
PubMed1992

Ontogeny of hepatic nuclear triiodothyronine receptor isoforms in the rat.

We have determined the contribution of the thyroid hormone receptor (TR) isoforms TR alpha 1 and TR beta 1 to the postnatal rise in rat hepatic nuclear T3-binding capacity. In agreement with previous studies, total hepatic nuclear binding capacity rose by about 8-fold from the 19th day of gestation to young adulthood at 2 months of age (0.10 +/- 0.03 to 0.86 +/- 0.17 pmol/mg DNA). The levels of specific TR species were measured by immunoprecipitation of T3-binding activity from hepatic extracts using a panel of antisera directed against specific regions of the TR isoforms. The difference between receptor immunoprecipitated with antibody against TR beta 1 and that precipitated with an antibody against an identical region in both TR beta 1 and TR alpha 1 was tentatively assumed to represent TR alpha 1. TR alpha 1 accounted for virtually all T3-binding activity in fetal liver on gestational day 19 (G19), increased by 2-fold shortly after birth, and remained constant thereafter. TR alpha 1 mRNA, on the other hand, was highest in concentration on G16 and fell by 50-75% in the adult. TR beta 1 was undetectable by immunoprecipitation of hepatic extracts from fetuses on G19. However, Northern analysis showed the presence of TR beta 1 mRNA in the fetal liver, which rose in concentration by 3- to 4-fold in late gestation and then remained constant. The contribution of TR beta 1 to total binding capacity rose to 33% and 40% on postnatal days 15 and 30, respectively, and to 80% in the adult liver. Immunohistochemical analyses of hepatic sections confirmed the presence of very low levels of TR beta 1 in fetal liver as early as G16 and G19, and a sharp rise in TR beta 1 protein concentration in the postnatal period. This indicated that the increase in TR beta 1-binding capacity results from increased TR beta 1 mass. The increase in TR beta 1-binding capacity, thus, is due to increased translational efficiency of the beta 1 mRNA or stabilization of the TR beta 1 protein. The prominence of TR alpha 1 in both rat fetal liver and fetal brain, as previously demonstrated in our laboratory, raises the possibility that this receptor isoform may carry out specialized functions in the fetus and that TR beta 1 subserves still other functions at later stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1992

Beta 1 isoform-specific regulation of a triiodothyronine-induced gene during cerebellar development.

Although tissue-specific expression of the alpha 1 and beta 1 thyroid hormone receptors (TR-alpha 1 and TR-beta 1) suggests isoform-specific function, transfection studies to date have failed to show consistent differences in their ability to regulate gene expression. We here provide evidence that TR-beta 1 but not TR-alpha 1 regulates the expression of the gene coding for PCP-2 in cerebellar Purkinje cells during neonatal rat development and that such regulation appears to be both T3 dependent and T3 independent. Examination of neonatal rats revealed that the levels of three mRNAs expressed in cerebellar Purkinje cells (myoinositol-1,4,5-triphosphate receptor, calbindin, and PCP-2) rise from neonatal day 1 to day 15. This rise is preceded by the previously documented surge in brain T3 and TR-beta 1. Methimazole-induced hypothyroidism sharply reduces, but does not abolish, the rise in these mRNAs. Concomitant T3 administration normalizes the process. In order to establish more directly the role of TR-beta 1 and T3, cotransfection experiments were performed in CHO cells with PCP-2-lacZ construct and TR isoforms. These studies showed that TR-beta 1, even in the absence of T3, regulated the expression of the transfected PCP-2 construct. T3 augments the response to TR-beta 1 alone by 40% (P < .01). TR-alpha 1 had no effect on PCP-2-lacZ expression either in the presence or absence of T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1991

Immunofluorescence localization of thyroid hormone receptor protein beta 1 and variant alpha 2 in selected tissues: cerebellar Purkinje cells as a model for beta 1 receptor-mediated developmental effects of thyroid hormone in brain.

Rat c-erbA beta 1 mRNA rises in cerebrum during the first 10 days of life, coincident with an increase in tissue triiodothyronine (T3) levels and T3-dependent brain development. These data suggest that the beta 1 receptor may mediate the T3 effect. However, in cerebellum c-erbA beta 1 mRNA levels were very low. Since cerebellar development, including dendritic arborization of Purkinje cells, is a T3-sensitive process, we assessed the levels of the beta 1 receptor protein in cerebellum during development. Antisera to unique peptide regions of beta 1 were raised. Their specificity was demonstrated by specific immunoprecipitation of the in vitro translated product, 85% immunoprecipitation of the T3 binding activity in hepatic nuclear extracts, and Western blot analysis of tissue extracts. Immunohistochemical studies using anti-beta 1 antiserum stained liver nuclei but not testis nuclei, which contain no T3 binding activity or beta 1 mRNA. In cerebellar Purkinje cells, an immunofluorescent signal, localized to the nucleus and more intense than that seen in the liver, was observed. A positive but weaker signal was also present in the granule cells. Thus, we may infer that the cerebellum contains significant concentrations of beta 1 receptor protein despite the low beta 1 mRNA content. Both the intensity of staining in Purkinje cell nuclei and immunoprecipitable beta 1 receptor binding capacity rose in the neonatal period. Antiserum to the non-T3 binding alpha 2 variant protein was also prepared and a distinctive pattern of fluorescence was observed. Strong fluorescence was seen in the nuclei of granule cells, but none was seen in Purkinje cells. The alpha 2 fluorescence in testis was high, consistent with the high levels of alpha 2 mRNA in this tissue. The fluorescent signal appeared to originate primarily in dividing spermatogonia. Our findings support the concept that the beta 1 receptor plays a central role in T3-induced brain development and strongly suggest that the Purkinje cell is a direct target for T3.

Amino Acid Sequence↗
PubMed1991

Prolonged fasting reduces rat hepatic beta 1 thyroid hormone receptor protein without changing the level of its messenger ribonucleic acid.

The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the beta 1-receptor (beta 1-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-beta 1-TR immunoglobulin G preparation. Although no changes in beta 1-TR mRNA concentration were noted, we observed a 60% fall in total binding capacity. beta 1-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in beta 1-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein.

Animals↗
PubMed1990

Relationship of c-erbA mRNA content to tissue triiodothyronine nuclear binding capacity and function in developing and adult rats.

We have quantitated in adult and developing rat tissues the molar concentrations of c-erbA alpha 1- and beta 1-mRNAs, which code for nuclear T3-binding proteins, and c-erbA alpha 2-mRNA, which is generated by alternate splicing of the alpha gene transcript and codes for a receptor variant that does not bind T3. Comparison of the concentrations of c-erbA alpha 1-mRNA, beta 1-mRNA, or their sum to the T3 nuclear binding capacity per mg of DNA in adult liver, kidney, heart, cerebrum, and cerebellum and during the ontogeny of liver and brain shows that the T3 binding capacity/c-erbA mRNA ratio is tissue-specific and related to developmental state. Administration of T3 resulted in a 40-50% fall in the alpha 1 signal of adult liver, kidney, and heart without changing either the beta 1 signal or T3 binding capacity. A 40-fold increase in rat brain beta 1-mRNA occurred in the transition between the 19-day gestational fetus and the 10-day-old neonate. This corresponds to the period during which the T3 content rises in brain and during which T3 is known to influence central nervous system development. Our findings indicate that important translational or post-translational factors influence nuclear binding capacity and raise the possibility that c-erbA beta 1 may play a primary role in mediating T3 effects in developing and adult animals.

Aging↗
PubMed1990

Binding of 3,5,3'-triiodothyronine (T3) and its analogs to the in vitro translational products of c-erbA protooncogenes: differences in the affinity of the alpha- and beta-forms for the acetic acid analog and failure of the human testis and kidney alpha-2 products to bind T3.

We have compared the affinities for T3 and the T3 analog binding characteristics of the in vitro translational products of seven c-erbA cDNAs (chicken c-erbA alpha; human placental c-erbA beta; rat c-erbA beta-1; rat c-erbA alpha-1; rat c-erbA alpha-2; human testis c-erbA alpha-2; and human kidney c-erbA alpha-2). Four of these (chicken c-erbA alpha, human placental c-erbA beta, rat c-erbA beta-1, rat c-erbA alpha-1) bound T3 with high affinity as previously described. When compared under identical conditions of synthesis and [125I]T3 binding, there was no significant difference between the affinity of the chicken c-erb A alpha-1 and the human c-erbA beta but in a more limited series the affinity of rat c-erbA beta-1 for T3 was 4.6-fold higher than that of the rat c-erbA alpha-1. In vitro translational products of the beta-probes showed a characteristic 2.2-fold higher triiodothyroacetic acid/T3 ratio than did the products of the alpha-probes, regardless of the species of origin of the probe. As previously established, the rat c-erbA alpha-2 product did not bind T3. However, in contrast to two published reports, the human testis and kidney alpha-2 probe products also failed to bind T3. These findings indicate that highly conserved C-terminal 37-40 residues are important for high affinity T3 binding by proteins encoded by the c-erb A family of genes.

Amino Acid Sequence↗
PubMed1989

Kinetics of induction by thyroid hormone of the two hepatic mRNAs coding for cytosolic malic enzyme in the hypothyroid and euthyroid states. Evidence against an obligatory role of S14 protein in malic enzyme gene expression.

In rat liver, triiodothyronine (T3) and dietary carbohydrate induce the expression of the genes coding for malic enzyme (ME) (EC 1.1.1.40) and S14 protein. The mRNAs for both ME and S14 are elevated under circumstances associated with augmented lipogenesis. Since the lag time in the induction of mRNA coding for S14 is short (20 min) and the lag time in the induction of the mRNA for ME is relatively long (2-6 h), the possibility arose that the induction of the ME gene by T3 was mediated by S14 protein. To test this hypothesis we examined the temporal relationship between the accumulation of the hepatic S14 protein and the mRNAs coding for ME. In confirmation of previous reports, we found that two mRNAs coded for ME, one 27 S and the other 21 S in size. The level of enzyme activity generated appeared to be determined by both mRNA species. Sequencing of the 27 S fragment established that this mRNA is generated as a consequence of the use of an alternate polyadenylation site downstream to that used in the 21 S mRNA. Unanticipated from the earlier descriptions was the finding of a markedly asynchronous response of these mRNAs to T3 in hypothyroid animals. The lag time following T3 administration was 90 min for the 27 S and fully 8-12 h for the smaller 21 S sequence. Despite the rapid rise of mRNA S14, the S14 protein could not be detected for approximately 12 h after T3 administration. This ruled out the possibility that S14 is an obligate mediator in the induction of the ME gene. A contrasting pattern was observed in the euthyroid state where both ME mRNAs had indistinguishable lag times of 2-3 h, and the S14 protein rose within the same time frame. The delayed response of the 21 S mRNA for malic enzyme in hypothyroid animals thus appears to be due to a reversible defect in the transcription of the ME gene.

Animals↗
PubMed1989

Time course of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and messenger ribonucleic acid, biliary lipid secretion, and hepatic cholesterol content in methimazole-treated hypothyroid and hypophysectomized rats after triiodothyronine administration: possible linkage of cholesterol synthesis to biliary secretion.

In an effort to define the mechanism by which thyroid hormone increases the synthesis of hepatic cholesterol, we have investigated both in hypophysectomized and methimazole-treated hypothyroid rats the time course of T3 effects on plasma cholesterol concentration, total hepatic cholesterol, the rate of biliary secretion of cholesterol, bile acids, and phospholipids, and the activity and mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, the rate-limiting enzyme in the hepatic synthesis of cholesterol. A single dose of 200 micrograms T3 was estimated to maintain at least 90% nuclear occupancy for the ensuing 54 h of the experiment. In both preparations the relative rise in biliary secretion of cholesterol exceeded that of other biliary constituents and preceded by 12 h an increase in HMG-CoA reductase enzyme activity and its mRNA. The level of total hepatic cholesterol remained constant throughout the experiment. We interpret these findings to suggest that T3-stimulated cholesterol synthesis is mediated by an antecedent T3-induced rise in biliary cholesterol secretion. We postulate that biliary cholesterol secretion is augmented by an intrahepatic shift of cholesterol and depletion of the hepatic sampling center responsible for the feedback regulation of cholesterol synthesis. The level of HMG CoA reductase mRNA appeared to govern enzyme activity in both preparations, but the ratio of mRNA to hepatic enzyme activity was substantially greater in the methimazole-treated compared with the hyphophysectomized animals.

Animals↗
PubMed1986

Dopamine autoreceptor regulation of the kinetic state of striatal tyrosine hydroxylase.

Tyrosine hydroxylase isolated from striatal synaptosomes exhibits biphasic Lineweaver-Burk kinetics for its tetrahydrobiopterin cofactor, consistent with multiple Km forms of the enzyme. Incubation of striatal synaptosomes with forskolin (EC50 0.45 microM) or dibutyryl cyclic AMP (EC50 1.2 mM), results in activation of tyrosine hydroxylase, isolated from these synaptosomes via conversion of the enzyme to a single low Km form (Km 40 microM). The activation of synaptosomal tyrosine hydroxylase by forskolin or dibutyryl cyclic AMP is not additive and is similar to activation seen with cyclic AMP-dependent protein kinase phosphorylation of purified tyrosine hydroxylase. The addition of dopamine (IC50 1.0 microM) (with nomifensine and pargyline) or apomorphine (IC50 30 nM) to the synaptosomal incubation medium blocks the activation of tyrosine hydroxylase by forskolin. This effect of dopamine and apomorphine can in turn be blocked by preincubation of the synaptosomes with the dopamine receptor antagonist haloperidol (IC50 30 nM and 4.5 nM, respectively) or chlorpromazine (IC50 50 nM versus apomorphine). In contrast to the forskolin data above, dopamine failed to block the activation of tyrosine hydroxylase by dibutyryl cyclic AMP. Addition of dopamine to the tyrosine hydroxylase assay, in amounts equivalent to that carried over from the synaptosomal incubation with the tyrosine hydroxylase, had no effect on forskolin-activated enzyme. The observations that dopamine and apomorphine can block forskolin activation of tyrosine hydroxylase, that this blockade can in turn be prevented by preincubation with haloperidol or chlorpromazine, and that the amount of dopamine required for blockade of forskolin activation in synaptosomes has no effect on tyrosine hydroxylase when added to the enzyme assay constitute the first clear evidence of a presynaptic dopamine receptor (autoreceptor). This autoreceptor regulates the activity of tyrosine hydroxylase by preventing or reversing cyclic AMP-dependent activation of the enzyme, probably through a decrease in the phosphorylation state of tyrosine hydroxylase. Failure of dopamine to block dibutyryl cyclic AMP activation of tyrosine hydroxylase suggests that, if forskolin and dibutyryl cyclic AMP activate tyrosine hydroxylase through identical changes in phosphorylation state, then autoreceptor regulation of tyrosine hydroxylase must occur through a decrease in cyclic AMP levels.

Adenylyl Cyclases↗