Human BM stem cells initiate angiogenesis in human islets in vitro.

BM stem cells may have regenerative effects on islet function through angiogenesis. Human islets (100islet equivalent/mL) were cultured alone (control) or co-cultured (experimental group) with whole human BM (1 × 10(6) cells/mL) for 210 days. A protein array measuring angiogenesis factors found upregulated (experimental vs control, day 210) proteins levels of VEGF-a (535 vs 2 pg/mL), PDGF (280.79 vs 0 pg/mL), KGF (939 vs 8 pg/mL), TIMP-1 (4592 vs 4332 pg/mL) and angiogenin (506 vs 97 pg/mL). Lower protein levels of angiopoietin-2 (5 vs 709 pg/mL) were observed. Depletion of pro-angiogenesis factors in co-culture decreased the effects of BM-induced islet vascularization. Depletion of VEGF-a, eKGF and PDGF significantly reduced islet vascularization but individual depletion of KGF and PDGF had less effects overall on vessel formation. BM-induced vascularization showed significant endothelial cell distribution. Islet vascularization was linked to islet growth. A decrease in islet size indicated poor vascularization. Insulin release was evident in the tissues generated from human islet-BM co-culture throughout the entire culture period. Significant increase in insulin (28.66-fold vs control) and glucagon (24.4-fold vs control) gene expression suggest BM can induce endocrine cell regeneration. In conclusion, BM promotes human islet tissue regeneration via regulation of angiogenesis factors.

Thyrotropin releasing hormone (TRH) may preserve pancreatic islet cell function: potential role in the treatment of diabetes mellitus.

Thyrotropin Releasing Hormone (TRH), first identified in the hypothalamus as a regulator of the Pituitary-Thyroid axis, has also been found in the beta-cell of the pancreas co-localised with insulin. The significance of this association is emphasised by the report that the TRH knock-out (KO) mouse is hyperglycemic. These findings have led to speculation that TRH may have a physiologic role in the regulation of carbohydrate metabolism. To understand better the role of TRH in the pancreas, TRH was administered to rats rendered diabetic from streptozotocin damage to the islets of Langerhans. This resulted in almost complete normalisation of the profound hyperglycemia. TRH is capable of reversing Diabetes Mellitus (DM) in an experimental animal model, possibly by promoting neogenesis of beta cells through induction of adult stem cells in the pancreas. These studies point to a potential therapeutic role for TRH in the treatment of DM in man.

Expression of thyrotropin-releasing hormone receptor in immortalized beta-cell lines and rat pancreas.

Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide, is expressed in pancreatic islets at peak levels during the late gestation and early neonate period. TRH increases insulin production in cultured beta-cells, suggesting that it might play a role in regulating pancreatic beta-cell function. However, there is limited information on TRH receptor expression in the pancreas. The aim of the present study was to explore the distribution of the TRH receptor in the pancreas and its function in pancreatic beta-cells. TRH receptor type 1 (TRHR1) gene expression was detected by RT-PCR and verified by Northern blotting and immunoblotting in the beta-cell lines, INS-1 and betaTC-6, and the rat pancreatic organ. The absence of TRH receptor type 2 expression in the tissue and cells indicated the tissue specificity of TRH receptor expression in the pancreas. The TRHR1 signals (detected by in situ hybridization) were distributed not only in islets but also in the surrounding areas of the pancreatic ductal and vasal epithelia. The apparent dissociation constant value for the affinity of [(3)H]3-methyl-histidine TRH (MeTRH) is 4.19 in INS-1 and 3.09 nM in betaTC-6. In addition, TRH induced epidermal growth factor (EGF) receptor phosphorylation with a half-maximum concentration of approximately 50 nM, whereas the high affinity analogue of TRH, MeTRH, was 1 nM. This suggested that the affinity of TRH ligands for the TRH receptor influences the activation of EGF receptor phosphorylation in betaTC-6 cells. Our observations suggested that the biological role of TRH in pancreatic beta-cells is via the activation of TRHR1. Further research is required to identify the role of TRHR1 in the pancreas aside from the islets.

[Analysis of heading time genotype for a rice male sterile line Zhenshan 97A].

Zhenshan 97A, a rice male sterile, were applied widely since the release of hybrid rice in 1973 in China, but the genotype of heading time in this sterile line was still unknown. This definitely limited the further use of this sterile line in breeding practice and re-production of hybrid seeds. To solve this problem, we analyzed the segregation pattern of phenotype of heading time in progenies from crosses between Zhenshan 97A and four tester cultivars, Akihikari (e1e1e2e2e3e3 Se-1eSe-1e), Koshihikari (E1E1E2E2e3e3Se-1eSe-1e), Nipponbare (E1E1e2e2e3e3Se-1Se-1) and Hinohikari (E1E1E2E2e3e3Se-1Se-1), whose genotypes of heading time were already known. The results showed that the genotype of heading time in Zhenshan 97A was e1e1e2e2E3E3Se-1Se-1 and it also carried a recessive inhibitor i-Se-1 for phtoperiod-sensitivity. Meanwhile, a major photoperiod-sensitive dominant genes Se-1 and other modified photoperiod-sensitive genes: i-Se-1, E3, Hd3(En-Se-1), Hd5 and Hd6, were identified in Zhenshan 97A by crossing with QTL nearly isogenic lines: NIL (Hd1), NIL (Hd2), NIL(Hd3), NIL(Hd5) and NIL(Hd6).

Effect of preproTRH antisense on thyrotropin-releasing hormone synthesis and viability of cultured rat diencephalic neurons.

To investigate a possible neurotropic role for thyrotropin-releasing hormone (TRH) in the central nervous system, we used recombinant antisense TRH adenovirus (TRHav) to "knock out" TRH in cultured 17-d fetal rat diencephalon. The morphology along with beta-galactosidase (beta-gal) enzyme histochemistry (X-gal staining) and TRH content (femtomoles/well) were used to measure the effect of antisense TRH virus. Control adenovirus mediated beta-gal transfection efficiency was nearly 85%, as shown by positive X-gal staining, and was without effect on cell morphology, TRH content, or the normal response to glucocorticoid (dexamethasone) exposure with enhanced TRH expression. A significant 90% decline in TRH content as well as changes in neuronal morphology (shrunken cell bodies and short dendrites) were observed after 14 but not 7 d following TRHav treatment. The addition of synthetic TRH peptide at 2.5 microM along with TRHav, but not dexamethasone, partly prevented the morphologic changes. No morphologic changes were seen in wild-type AtT20 cells, a pituitary cell line that does not produce TRH. To investigate whether neuronal death from loss of proTRH was owing to apoptosis, neuronal DNA change by means of fluorescent dye H-33342 staining, TUNEL staining, and DNA laddering analysis was examined. Eighty to 90% positive H-33342 and TUNEL staining as well as a 180- to 200-bp DNA fragment on DNA laddering analysis were found as compared to control. These results indicate that proTRH gene expression prevents neuronal apoptosis and may play a role in neuronal development and function.

Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting.

Because cocaine- and amphetamine-regulated transcript (CART) coexists with alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus neurons and we have recently demonstrated that alpha-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNA-containing neurons and CART- and alpha-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all alpha-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for alpha-MSH. In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in approximately 80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in <10% of the anterior parvocellular subdivision neurons. In addition, >80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.

Evidence from studies with N-ethyl-maleimide and 12-O-tetradecanoylphorbol-13-acetate that AP-1 and CREB are involved in the glucocorticoid activation of TRH gene expression in hypothalamic cultures.

To determine whether c-fos/c-jun (AP-1) and CREB mediate glucocorticoid stimulated TRH gene regulation, we investigated the effect of N-ethyl-maleimide (NEM), an alkylating agent, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on this process. NEM decreased while TPA increased TRH levels in rat hypothalamic culture, changes similar to their effects on CREB and Fos/Jun proteins in the AtT20 cell line. This suggests that glucocorticoid stimulation of TRH gene expression may be regulated by the AP-1 complex and CREB pathway.

Advantage of double labeled in situ hybridization for detecting the effects of glucocorticoids on the mRNAs of protooncogenes and neural peptides (TRH) in cultured hypothalamic neurons.

In order to study the effect of glucocorticoids on thyrotropin-releasing hormone (TRH) and protooncogenes, we describe a double labeled in situ hybridization method to explore this issue. The development of non-isotopic in situ hybridization histochemistry has proven to be an important tool for cellular and molecular studies in neurobiology [C.L.E. Moine, E. Normand, B. Bloch, Use of non-radioactive probes for mRNA detection by in situ hybridization: interests and applications in the central nervous system, Cell. Mol. Biol. 41 (1995) 917-923]. These methods involve the anatomic localization of labeled RNA or DNA molecules which hybridize with complementary target RNA or DNA sequences in the cell. With regard to gene expression, in situ hybridization allows the study of specific mRNA levels and the distribution between various cell types. It also allows the comparison of mRNA levels at various stages of development. Double labeled in situ hybridization is able to detect the colocalization of two different mRNAs simultaneously. Accordingly, this approach is utilized for specific studies involving the expression and distribution of TRH mRNA and the protooncogenes, c-fos/c-jun, in cultured rat hypothalamic neurons [L. G. Luo, I.M.D. Jackson, Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons, Brain Research 791 (1998) 56-62]. Our protocol for double labeled in situ hybridization reflects a modification of a number of original protocols developed by others [H. Breitschopf, G. Suchanek, R.M. Gould, D.R. Colman, H. Lassmann, In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain, Acta Neurropathol. 84 (1992) 581-587; S. McQuaid, J. McMahon, G.M. Allan, A comparison of digoxigenin and biotin labeled DNA and RNA probes for in situ hybridization, Biotech. Histochem. 70 (1995) 147-154; E. Hrabovszky, M.E. Vrontakis, S.L. Petersen, Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats, J. Histochem. Cytochem. 43 (1995) 363-370]. This technique can be readily applied to various studies of cellular gene expression in the mammalian nervous system involving other neural peptides and transcription factors.

Antidepressants inhibit the glucocorticoid stimulation of thyrotropin releasing hormone expression in cultured hypothalamic neurons.

BACKGROUND: The pituitary thyroid axis is frequently effected in human depression possibly due to alteration in hypothalamic thyrotropin releasing hormone (TRH) secretion. Since clinical recovery is associated with normalization of thyroid function, the direct effect of antidepressants on TRH expression in a well established fetal rat hypothalamic neuronal culture system was investigated. METHODS: Fetal rat hypothalamic neurons (day 17) in culture were treated with different concentrations of antidepressants with or without glucocorticoids for 7 days following which TRH content was measured by radioimmunoassay (RIA). RESULTS: The results showed that Imipramine (IMIP), a tricyclic antidepressant (TCA), decreased the TRH content in a dose-dependent manner (from 80.7 +/- 4.9, at 10(-9) mol/L, to 14.1 +/- 0.6, at 10(-5) mol/L, fmol/well; P < 0.05). Desipramine (DESI), another tricyclic antidepressant, also decreased the TRH content (from 63.6 +/- 2.5, at 10(-9) mol/L, to 12.6 +/- 0.4, at 10(-5) mol/L, fmol/well; P < 0.05). Sertraline (SERT) and Fluoxetine (FLUO), serotonin selective reuptake inhibitors (SSRI), also decreased TRH content in a dose dependent manner (from 83.9 +/- 7.9, at 10(-10) mol/L, to 7.6 +/- 0.4, at 10(-5) mol/L, and from 41.66 +/- 2.5, at 10(-8) mol/L, to 17.54 +/- 0.92, at 10(-6) mol/L, fmol/well, respectively; both P < 0.05). We then tested the effect of these antidepressants on the Dex stimulation of TRH content. IMIP, DESIP and FLUO at 10(-6) mol/L reduced the TRH response to glucocorticoid stimulation (36.4 +/- 4.0, 56.6 +/- 2.4, 23.75 +/- 4.0, respectively vs 107 +/- 7.5 fmol/well; P < 0.05). CONCLUSION: This raises the possibility that the enhanced thyroid function in depression, which we postulate, may result in part from glucocorticoid stimulation of TRH gene expression, can be reversed by antidepressants through a direct effect on the TRH neuron. However, other mechanisms may need to be invoked in addition since basal TRH content was also reduced.

Antisense oligomers of cfos and cjun block glucocorticoid stimulation of thyrotropin-releasing hormone (TRH) gene expression in cultured anterior pituitary cells.

The proto-oncogenes, cfos/cjun, are co-localized with thyrotropin-releasing hormone (TRH) in cultured anterior pituitary cells and increase following exposure to dexamethasone (Dex). To assess the role of cfos and cjun in the Dex stimulation of TRH gene expression, we used antisense oligonucleotides to block cfos and cjun expression in order to reduce formation of activating protein-1 (AP-1). The results showed that the antisense oligonucleotides together effectively reduced cfos/cjun gene expression and consequently the glucocorticoid stimulation of TRH peptide and mRNA. The findings indicate that cfos/cjun are involved in the glucocorticoid activation of TRH gene expression.

Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons.

To explore whether the protooncogenes, c-fos/c-jun, might be involved in regulating the effect of glucocorticoids on thyrotropin-releasing hormone in fetal rat diencephalic neurons, their localization and transcriptional activity were investigated using double-labeled in situ hybridization, Northern blot and nuclear run-on assays. The results showed that TRH mRNA was coexpressed with both c-jun and c-fos in the same neurons. Treatment with dexamethasone, a synthetic glucocorticoid, at 10(-8) M, enhanced transcriptional activity resulting in an increase in both cell number and intensity of all three mRNAs. The existence of c-fos/c-jun in thyrotropin-releasing hormone neurons and the increased transcriptional activity following dexamethasone treatment suggests that these protooncogenes could mediate the effect of glucocorticoids on thyrotropin-releasing hormone gene expression.

Identification of the thyrotropin-releasing hormone precursor, its processing products, and its coexpression with convertase 1 in primary cultures of hypothalamic neurons: anatomic distribution of PC1 and PC2.

The processing of pro-TRH, has been extensively studied in our laboratory using a corticotropic cell line, AtT20, transfected with the pro-TRH gene. We have also demonstrated that the convertases PC1 and PC2 process pro-TRH to cryptic peptides in vitro. However, although these processing pathways have been well characterized in vitro, little is known about the processing and subcellular distribution of pro-TRH and its derived peptides in hypothalamic neurons, an endogenous source of pro-TRH and PC enzymes. In this study we used multiple approaches to identify, both biochemically and anatomically, the presence and localization of pro-TRH (26 kDa) and its processing products. We also investigated the presence of PC1 and PC2 enzymes and the coexpression of pro-TRH and PC1 messenger RNAs. Identification of the TRH precursor was demonstrated by 1) Western blot analysis of cellular extracts, 2) immunoprecipitation of radiolabeled pro-TRH followed by analysis on acrylamide gel electrophoresis, 3) fluorescence immunocytochemistry, and 4) immunoelectron microscopy. The presence of the convertases PC1 and PC2 was determined by Western blot analysis of cellular extracts and fluorescence immunocytochemistry. The coexpression of pro-TRH with PC1 was shown by double in situ hybridization. Our findings support three main conclusions. First, this primary culture system of hypothalamic neurons is suitable for characterizing pro-TRH processing as well as identifying the anatomical location of its processing products. Second, prohormome processing takes place during axonal transport after removal of the signal peptide in the endoplasmic reticulum, and subsequent cleavages of the prohormone occur as intermediate peptides move down the axon toward the nerve terminal. This coupled transport-processing phenomenon may provide the necessary mechanism to ensure flexibility in differential processing of specific protein sequences that are determined by the secretory needs of cells. It appears that certain intermediate peptides differ in their subcompartmental distribution, suggesting the possibility of a differential processing and maturation of pro-TRH-derived peptides. Thirdly, the 87-kDa form of PC 1 may initiate the processing of pro-TRH at the Golgi complex level, which then continues to be processed by PC1 and PC2 in later stages of the secretory pathway.

Activation of thyrotropin-releasing hormone gene expression in cultured fetal diencephalic neurons by differentiating agents.

The present studies were undertaken to investigate the effect of 5-bromo-2'-deoxyuridine (BrdU; 50 microM) or forskolin/3-isobutyl-1-methylxanthine (F/I; 10/500 microM) on TRH gene expression in cultured fetal diencephalic cells. BrdU as well as drugs such as F/I that raise intracellular cAMP levels had been previously termed differentiating agents because they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalons may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracellular TRH and pro-TRH messenger RNA concentrations in cultured diencephalic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to cause maintenance of high TRH levels after withdrawal of the drug, F/I had to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expression in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I exposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulation of somatostatin and arginine vasopressin, which were determined in parallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these and possibly other neuropeptides in vitro.

Glucocorticoids stimulate thyrotropin-releasing hormone gene expression in cultured hypothalamic neurons.

Although there is much evidence indicating that glucocorticoids (GC) inhibit the hypothalamic-pituitary-thyroid axis in both rat and man in vivo, there have been no previous studies on the direct effect of GC on hypothalamic TRH neurons in vitro. In this laboratory, we developed fetal rat (day 17) diencephalic neuronal cultures in the presence of 5'-bromo-2-deoxyuridine, a cell-differentiating agent that stimulates TRH gene expression. In 12 separate experiments, dexamethasone (Dex) induced a 2.2-fold increase in TRH content vs. the control value (P < 0.01). Dex (10(-8)M) enhanced TRH messenger RNA (mRNA) 1.6-fold (n = 75 wells; P < 0.01) by nonisotopic in situ hybridization. On Northern blot analysis using a 32P-labeled complementary RNA probe, TRH mRNA was enhanced 3-fold (n = 4; P < 0.01). Nuclear run-on analysis revealed that Dex enhanced transcription 7.7 fold (n = 3; P < 0.01). We conclude that 1) Dex stimulates the expression of TRH peptide and TRH mRNA in cultured hypothalamic neurons; 2) the increase in TRH mRNA results (at least in part) from enhanced transcription; and 3) the reported in vivo depression of TRH in the paraventricular nucleus after GC stimulation suggests that this effect must be mediated indirectly on the TRH neuron.

Effect of nitroprusside (nitric oxide) on endogenous dopamine release from rat striatal slices.

It is becoming apparent that the synthesis of nitric oxide (NO) from L-arginine not only explains endothelium-dependent vascular relaxation, but is a widespread mechanism for the regulation of cell function and communication. We examined the role of NO on the endogenous dopamine (DA) release from rat striatum. Nitroprusside, in the concentration range of 3-100 microM, induced a dose-dependent increase in the endogenous DA release from rat striatal slices. The maximal response was 330% over the baseline release. A higher concentration of nitroprusside (300 microM) produced an inhibitory effect on the spontaneous release of DA. L-Arginine (10 and 100 microM), a substrate in the NO-forming enzyme system, also produced an elevation of DA release. L-Arginine-induced DA release was attenuated by NG-monomethyl-L-arginine, an inhibitor of NO synthase. NADPH (1 microM), a cofactor of NO synthase, enhanced L-arginine-induced DA release. These results suggest a possible involvement of NO in the DA release process in rat striatum.

[Effect of opioid peptides on progesterone production by rat luteal cells in vitro].

The effect of exogenous opioid peptides on progesterone production by incubated rat luteal cells was studied. beta-endorphin (beta-EP) stimulated progesterone production in a dose-dependant manner (10(-8)-10(-6) mol/L); dynorphin exhibited a stimulatory effect only at 10(-6) mol/L, while Met-enkephalin had no substantial effect at dose from 10(-10)-10(-6) mol/L. mu-opioid receptor agonists DAGO and morphine also stimulated progesterone production. The stimulatory actions of beta-EP, DAGO and morphine were reversed completely by naloxone. On account of the fact that the beta-EP level in rat plasma is lower than that in the ovary, it seemed that beta-EP may be an intra-ovarian luteotrophic factor and be involved in the regulation of progesterone production. This action of beta-EP may be mediated by mu-opioid subtype receptors.

[A study of the regulatory role of GABA on the LHRH release in the median eminence of rats].

The present study was undertaken to observe the effect of GABA and NA on LHRH neuronal terminals in the median eminence (ME). The results showed that GABA (10(-6) mol/L) significantly increased LHRH and NA release from ME, i.e., respectively from 27.3 +/- 2.5 pg/100 microliters to 150.4 +/- 27.9 pg/100 microliters and from 50.9 +/- 4.2 pg/100 microliters to 105.5 +/- 19.1 pg/100 microliters (both P less than 0.01 vs control group). These effects of GABA could be blocked by bicuculline. When bicuculline and GABA (10(-6) mol/L) were added into the ME, the secretion of LHRH decreased to 18.2 +/- 1.9 pg/100 microliters and that of NA to 43.9 +/- 3.4 pg/100 microliters. The effect of GABA on LHRH release may be mediated by NA. When endogenous NA was depleted by reserpine, GABA increased LHRH secretion only by 26.5% instead of 451.9% as in the normal animal.

l-tetrahydropalmatine increases leucine enkephalin levels in corpus striatum of rats.

The effect of chronic l-tetrahydropalmatine (l-THP) administration on the level of leucine enkephalin (Leu-Enk) in rat corpus striatum was studied. After l-THP sc injection once daily for 2 wk, the striatal Leu-Enk level was elevated dose-dependently. However, a single injection of l-THP failed to change the Leu-Enk level. When rats received sc Sch-23390, a selective D1 antagonist, 15 nmol.kg-1 tid for 2 wk, striatal content of Leu-Enk increased from 0.17 +/- SD 0.03 ng.mg-1 tissue in control group to 0.23 +/- SD 0.05 ng.mg-1 tissue in Sch-23390 group (n = 8, P less than 0.05). Sulpiride (Sul), a selective D2 antagonist, 140 mumol.kg-1 sc given bid for 2 wk had no significant effect on the striatal Leu-Enk content. The results suggested that the blockade of D1 receptors by l-THP might be responsible for the increase of the striatal Leu-Enk content in rat.