Identification and Localization of Insulin-Like Growth Factor Receptors in Human Infant Pituitary Gland—Similar Distribution to Somatotrophs

Insulin-like growth factors(IGF) are involved in feedback regulation of growth hormone(GH) secretion from the pituitary. Though receptors for IGF-I and IGF-II have been identified on particulate preparations of rat pituitary, their localization and relationship to GH-secreting acidophils has not been determined. We used quantitative in vitro autoradiography and immunocytochemistry to simultaneously determine the distribution of IGF receptors and GH-secreting cells in human infant pituitary gland. Frozen or fixed post-mortem human infant pituitary glands were sectioned for binding studies, or immunocytochemistry. Binding for IGF-I and IGF-II showed characteristic specificity for respectively Type I and Type II receptors. Binding sites were visualized throughout the pituitary gland, with similar density and distribution for IGF-I and IGF-II receptor sites. Receptor density was two-fold higher in anterior than posterior pituitary, with highest density in the lateral horns of the anterior pituitary. The distribution of GH-containing cells (acidophils) was similar to IGF receptor distribution. Increased density of IGF receptors in regions of GH-secreting cells may point to the mechanism whereby IGF uniquely inhibits synthesis of human GH in contrast to its promotion of synthetic processes in other cells.     

Binding Sites for Vasopressin in the Human Pituitary are Associated with Corticotrophs and may Differ from Other Known Vasopressin Receptors

In view of the fact that Vasopressin can induce pituitary adrenocorticotrophin release, we performed an autoradiographical study of [³H]arginine vasopressin binding in human pituitary tissue obtained post-mortem from adults and foetuses. Sites of specific, high affinity binding (IC₅₀ 3 to 5 nM) were detected as patches in the anterior lobe and at the junction between the anterior and neural lobes. The neural lobe was not labelled. Immunocytochemical studies performed on human pituitary tissue showed that [³H]arginine vasopressin only marked zones which correspond to areas rich in cells immunoreactive to adrenocorticotrophin. We conclude that in the human pituitary, corticotrophs bear vasopressin binding sites. Since non-radioactive synthetic structural analogues of vasopressin acting as V₁ and V₂ agonists or antagonists failed to compete for binding of radioligand in the human pituitary, while a V₁-type agonist displaced [³H]arginine vasopressin binding in the rat pituitary, we postulate that binding sites in the human pituitary may differ from the previously known vasopressin receptors.     

Evidence For Two Neurochemical Divisions in the Human Nucleus Accumbens

The neurochemical anatomy of the human nucleus accumbens was studied by comparing the distributional patterns of [³H]DAMGE (μ opioid receptor), [³H]bremazocine (κ opioid receptor), [³H]SCH-23390 (D₁-like dopamine receptor), [³H]7-OH-DPAT (D₃ dopamine receptor) binding, preproenkephalin mRNA and acetylcholinesterase activity in sections of post mortem human striatum. Our results demonstrate the presence of at least two neurochemically distinct divisions within the human nucleus accumbens, which may be homologous to the 'shell'and'core'divisions of the nucleus as found in the rat.     

Distribution of Somatostatin Receptors 1, 2 and 3 mRNA in Rat Brain and Pituitary

In this study sequence-specific antisense oligonucleotide probes have been used to investigate the distribution of the mRNAs coding for the somatostatin receptor subtypes termed somatostatin receptor 1, somatostatin receptor 2 and somatostatin receptor 3 in the rat brain and pituitary using in situ hybridization techniques. The three receptor subtype mRNAs were found to be widely distributed in the brain with different patterns of expression, but with some overlap. Somatostatin receptor 1 mRNA was particularly concentrated in the cerebral and piriform cortex, magnocellular preoptic nucleus, hypothalamus, amygdala, hippocampus, and several nuclei of the brainstem. Somatostatin receptor 3 mRNA was very abundant in the cerebellum and pituitary (in contrast to somatostatin receptor 1), but it was also found in hippocampus, amygdala, hypothalamus and in motor nuclei of the brainstem. Somatostatin receptor 2 mRNA levels were very low relative to the other two mRNAs evaluated. Receptor 2 mRNA was observed in the anterior pituitary, and in the brain it was found in the medial habenular nucleus, claustrum, endopiriform nucleus, hippocampus, some amygdala nuclei, cerebral cortex and hypothalamus. None of the three somatostatin receptor mRNAs studied here was found in the caudate nucleus. Northern analysis revealed distinct sizes of mRNAs for each subtype, and displacement experiments showed that each probe sequence was subtype-specific.     

Opioid system functional regulation in neurological disease management

There is increasing evidence to suggest a role for the opioid system in the control of pathophysiology of neurological disorders (Alzheimer's, Parkinson's, and Huntington's diseases, spinal cord injury, epilepsy, hypoxia, and autism). Resuscitation of the altered expression of the opioid system in various neurological disorders is of therapeutic importance. Such treatment may be beneficial in ameliorating the clinical symptoms of the disorder. This Mini‐Review provides a brief update on opioid system regulation in neurological disorders and focuses on the opioids' pharmacological importance. © 2010 Wiley‐Liss, Inc.     

Temperature and Protein Kinase C Modulation of Gonadotropin-Releasing Hormone Receptors in Pituitary Cells from Intact or Castrated Male Rats

Binding constants of [¹²⁵ I]Des-Gly₁₀-(D-Ala₆)-gonadotropin-releasing hormone-ethylamide (GnRHa) to dispersed pituitary cells were evaluated in a 4-day culture. Cells were sampled either from intact or from castrated male rats and binding was measured at various temperatures before or after treatment with phorbol-12-myristate-13-acetate (PMA) or 1-5-(isoquinolinyl-sulfoxyl) 2-methylpiperazine (H7), which activate or inhibit, respectively, protein kinase C (PKC). In cells from intact rats incubated with increasing concentrations of ligand at 21 °C for 25 min, the Scatchard plot was not linear and calculation of the Hill coefficient (N_H) was indicative of positive cooperativity (N_H= 1.26 ± 0.02). Such non-linearity was not observed when cells were incubated at 0.5 °C for 3 h. In that condition the maximal number of binding sites measured at equilibrium (B_max) increased (15.1 ± 0.05 versus 9.3 ± 0.5 fmoles × mg^?1 proteins at 21 °C). Two control experiments permitted us to rule out the possibility that lower B_max at 21 °C might reflect internalization: 1) Cells were first incubated with the ligand at 21 °C for 25 min and subsequently for 3 additional hours at 0.5 °C. Preincubation did not affect the B_max obtained at 0.5 °C; 2) when the radioligand bound to the cell surface was washed out with an acidic buffer, only 13% of the specific radioactivity was retained irrespective of the ligand concentration applied, a much lower value than the 40% binding difference observed between 0.5°C and 21°C. When the cells were incubated with PMA, the Scatchard plot was linearized and the B_max recorded at 21 °C increased by 50% over control cells (13 ± 0.7 fmoles × mg^?1 proteins). Conversely, inhibition of PKC by H7, a preferential PKC inhibitor, was ineffective. In contrast, cells sampled from castrates exhibited linear and comparable Scatchard plots at either 0.5 ° or 21 °C, with B_max values of 14.4 ± 0.3 and 15 ± 0.34 fmoles × mg^?1 proteins, respectively. PKC activation did not affect binding in that model, but H7 decreased the number of sites (B_max= 10.7 ± 0.9) and induced appearance of positive cooperativity (N_H= 1.36 ± 0.07). Taken together, these experiments reveal a pool of GnRH receptors in the pituitary of intact rats that recognizes the ligand in a phosphorylation-dependent manner. These binding sites also became evident when biological properties of the membrane were modified by temperature or cell homogenization. After castration, PKC activation was no longer a prerequisite for recruitment of the total population of receptors whereas protein kinase inhibition resulted in a reduction of maximal binding. Finally, our observations demonstrate that GnRH binding can exhibit positive cooperativity, either on normal cells or on cells from castrates after protein kinase inhibition, suggesting that such a cooperativity is not related to a GnRH-dependent phosphorylation.     

Neuroanatomical and Physiological Evidence for the Involvement of Pituitary Adenylate Cyclase-Activating Polypeptide in the Regulation of the Distal Lobe of the Frog Pituitary

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino-acid peptide which belongs to the glucagon/secretin/ vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ridibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP-containing neurons and fibers was examined by the indirect immunofluores-cence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose-dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED₅₀= 2 × 10^?8 M). At the maximum dose tested (5 × 10^?6 M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production. In contrast, the truncated form [Des-His¹frog PACAP38 did not affect adenylate cyclase activity demonstrating therefore that the N-terminal histidyl residue is essential for the biological activity of the peptide. [Des-His¹]frog PACAP38 did not antagonize the stimulatory effect of frog PACAP38 or PACAP27 on cAMP production. Taken together, these data support the concept that, in amphibians as in mammals, PACAP may act as a hypophysiotropic neuropeptide.     

Characterization of the GABA-lnduced Current in Frog Pituitary Melanotrophs

The molecular mechanisms regulating GABA_A receptor activity in cultured frog melanotrophs were studied using the patch-clamp technique. In the whole-cell configuration, application of GABA evoked a dose-related increase of inward chloride currents. The ED₅₀ value, estimated from the sigmoidal dose-response curve was 2 × 10^?6 M and the Hill coefficient was 1.55. The amplitude of the GABA-induced current decayed with time. Kinetics analysis of the desensitization revealed that the time-course of the current decrement was fitted by one exponential. Graded doses of GABA or association of GABA with the benzodiazepine receptor agonist flunitrazepam accelerated the desensitization process. In contrast, the time-course of the current did not significantly vary at different holding potentials. In the outside-out configuration, GABA was found to activate channels which displayed three unitary conductance levels (8, 15 and 30 pS). The channel openings of the more frequent conductance level (30 pS) exhibited short and long lasting open states (1.2 and 28.3ms at -60mV). Altogether these data reveal that frog melanotrophs possess a single population of GABA_A receptors which interconvert into a higher affinity state in the presence of benzodiazepine receptor agonists. Two GABA molecules must bind to the receptor to trigger long lasting channel openings. In addition, the activity of the GABA_A receptor appears to be independent of the accumulation of intracellular chloride ions.     

环孢素A影响人胎垂体细胞免疫原性的研究

本文将胎垂体细胞分别放入含与不含1.0μg/ml环孢素A(CsA)的两种培养液中培养2周,经免疫电镜检查及培养的细胞免疫家兔测定兔血清中抗人胎脑抗体效价的方法,证实在含CsA培养液中培养的细胞的免疫原性,较同期在不含CsA培养液中培养的细胞的免疫原性明显降低。结果提示:用CsA预处理胎垂体细胞,可显著降低其免疫原性。     

Sexual Dimorphism in the Distribution of a-Neoendorphin-Like Immunoreactivity in the Anterior Pituitary of the Rat

The localization of the opioid peptide α-neoendorphin (α-Neo-E) was studied in the anterior pituitary of normal and castrated male and normal female rats, Immunoreactive (ir) cells were noted in both sexes. These α-Neo-E-ir cells were further characterized using double immunostaining with an elution-restaining procedure. It was seen that in males, α-Neo-E-ir cells corresponded mainly to luteinizing hormone/follicle-stimulating hormone cells and a few thyroid-stimulating hormone (TSH) cells, whereas in females, virtually all α-Neo-E-ir cells corresponded to TSH cells. Castration of male rats caused, within 3 to 5 days, a dramatic decrease in the number of α-Neo-E-ir gonadotrophs, whereas the number of a-Neo-E-ir TSH cells tended to increase. Two weeks after castration, however, most α-Neo-E-ir cells were also follicle-stimulating hormone-ir. This study demonstrates that in the anterior lobe of the rat, α-Neo-E-ir is located within gonadotrophs and/or thyrotrophs, depending on the sex. In addition, results obtained following castration suggest that the expression of this peptide in the anterior pituitary depends upon the steroid environment, possibly indicating that α-Neo-E is implicated in the regulation of the pituitary-gonadal axis.     

垂体瘤转化基因在垂体大腺瘤中表达的研究

目的 探讨垂体瘤转化基因（PTTG）在垂体大腺瘤中的表达及意义。方法收集经手术和病理证实的垂体大腺瘤患者40例，其中无功能腺瘤22例，生长激素（GH）腺瘤8例，泌乳素（PRL）腺瘤10例。同期经手术和病理证实的垂体微腺瘤11例为对照组，其中促肾上腺皮质激素（ACTH）腺瘤8例。PRL腺瘤3例。采用免疫组化技术（LSAB法）检测垂体大腺瘤和垂体微腺瘤中PTTG的表达水平。结合临床资料及影像学分级标准，分析PTTG表达水平与垂体大腺瘤发生机制、生物学行为之间的联系。结果40例垂体大腺瘤中均发现PTTG的表达显著高于垂体微腺瘤组（P〈0．01）。在侵袭性垂体大腺瘤中，PTTG的表达显著高于非侵袭性垂体大腺瘤（P〈0．01）。PTTG的表达与大腺瘤向鞍上生长的高度和向海绵窦侵袭性生长的程度显著相关（P〈0.05）。结论PTTG表达增高与垂体大腺瘤的生长以及肿瘤的侵袭性密切相关。PTTG的表达水平及结合影像学资料可以为患者预后及术后的辅助治疗提供可靠的判断依据。     

Mechanism of Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Human Nonfunctioning Pituitary Tumors

Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca²⁺]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca²⁺]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 ± 10 to 214 ± 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 ± 5.4 to 73.2 ± 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6–27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca²⁺]i rise in cells obtained from 7 NFPA (from 110 ± 34 to 151 ± 40 nM [Ca²⁺]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 μM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca²⁺ as well as blockade of L-type Ca²⁺ channels by dihydropyridine antagonists abolished [Ca²⁺]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.     

CD105在垂体腺瘤中的表达及其意义

目的 探讨血管内皮标记物CD105在人脑垂体腺瘤中的表达及其意义.方法 免疫组化SP法分别以CD34、CD105为指标检测28例人脑垂体瘤中的微血管密度(MVD),分析其与肿瘤侵袭性的关系及两者的特异性.结果 CD34、CD105在非侵袭性垂体瘤中的表达强度即MVD分别为(36.21±19.96)、(17.61±15.51),在侵袭性垂体瘤中的表达强度MVD为(48.59±16.53)、(21.92±14.83).2者的表达强度均与肿瘤的侵袭性无关(t=0.184, P＞0.05,及t=0.632, P＞0.05).CD105无论在非侵袭性垂体瘤或侵袭性垂体瘤中的表达强度均明显低于CD34(t=1.63,P＜0.05及t=4.92,P＜0.01).结论 MVD与垂体瘤的侵袭性无关.作为一种新生血管内皮标记物,CD105较泛血管内皮标记物CD34具有明显的特异性,是一种更好的MVD计数指标.     

Opioid peptide receptor studies. 16. Chronic morphine alters G-protein function in cells expressing the cloned mu opioid receptor

Chronic morphine treatment results in functional uncoupling of the mu opioid receptor and its G protein in both cell culture and animal models. In the present study, Chinese hamster ovary (CHO) cells stably expressing the cloned human mu opioid receptor (hMOR-CHO cells) were incubated with 1 microM of morphine (or no drug) for 20 h. Subsequently, we assessed DAMGO- and morphine-stimulated [(35)S]-GTP-gamma-S binding and agonist-mediated inhibition of forskolin-stimulated cAMP accumulation. Using a single concentration of [(35)S]-GTP-gamma-S (0.05 nM), chronic morphine treatment did not significantly change basal [(35)S]-GTP-gamma-S binding, shifted the morphine EC(50) from 59 nM to 146 nM, and decreased the maximal stimulation (E(max)) from 201% to 177%. Similar results were observed with DAMGO. Binding surface analysis resolved two [(35)S]-GTP-gamma-S binding sites (high-affinity and low-affinity sites). In control cells, morphine stimulated [(35)S]-GTP-gamma-S binding by increasing the B(max) of the high-affinity site. In morphine-treated cells, morphine stimulated [(35)S]-GTP-gamma-S binding by decreasing the high-affinity K(d) without changing the B(max). Morphine treatment increased the EC(50) (5-11-fold) for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation. These changes were not observed in cells expressing a mutant mu opioid receptor which does not develop morphine tolerance, suggesting that the changes in [(35)S]-GTP-gamma-S binding observed in hMOR-CHO cells result from the development of morphine tolerance.     

HDAC6 Inhibition Reverses Cisplatin-Induced Mechanical Hypersensitivity via Tonic Delta Opioid Receptor Signaling

Peripheral neuropathic pain induced by the chemotherapeutic cisplatin can persist for months to years after treatment. Histone deacetylase 6 (HDAC6) inhibitors have therapeutic potential for cisplatin-induced neuropathic pain since they persistently reverse mechanical hypersensitivity and spontaneous pain in rodent models. Here, we investigated the mechanisms underlying reversal of mechanical hypersensitivity in male and female mice by a 2 week treatment with an HDAC6 inhibitor, administered 3 d after the last dose of cisplatin. Mechanical hypersensitivity in animals of both sexes treated with the HDAC6 inhibitor was temporarily reinstated by a single injection of the neutral opioid receptor antagonist 6β-naltrexol or the peripherally restricted opioid receptor antagonist naloxone methiodide. These results suggest that tonic peripheral opioid ligand-receptor signaling mediates reversal of cisplatin-induced mechanical hypersensitivity after treatment with an HDAC6 inhibitor. Pointing to a specific role for δ opioid receptors (DORs), Oprd1 expression was decreased in DRG neurons following cisplatin administration, but normalized after treatment with an HDAC6 inhibitor. Mechanical hypersensitivity was temporarily reinstated in both sexes by a single injection of the DOR antagonist naltrindole. Consistently, HDAC6 inhibition failed to reverse cisplatin-induced hypersensitivity when DORs were genetically deleted from advillin⁺ neurons. Mechanical hypersensitivity was also temporarily reinstated in both sexes by a single injection of a neutralizing antibody against the DOR ligand met-enkephalin. In conclusion, we reveal that treatment with an HDAC6 inhibitor induces tonic enkephalin-DOR signaling in peripheral sensory neurons to suppress mechanical hypersensitivity.SIGNIFICANCE STATEMENT Over one-fourth of cancer survivors suffer from intractable painful chemotherapy-induced peripheral neuropathy (CIPN), which can last for months to years after treatment ends. HDAC6 inhibition is a novel strategy to reverse CIPN without negatively interfering with tumor growth, but the mechanisms responsible for persistent reversal are not well understood. We built on evidence that the endogenous opioid system contributes to the spontaneous, apparent resolution of pain caused by nerve damage or inflammation, referred to as latent sensitization. We show that blocking the δ opioid receptor or its ligand enkephalin unmasks CIPN in mice treated with an HDAC6 inhibitor (latent sensitization). Our work provides insight into the mechanisms by which treatment with an HDAC6 inhibitor apparently reverses CIPN.     

人体血浆和垂体腺瘤组织中的神经肽Y含量及其临床意义

目的 :研究垂体腺瘤患者血浆和肿瘤组织中的神经肽Y(NPY)浓度与垂体腺瘤的关系。方法 :放射免疫分析方法测定 3 3例垂体腺瘤组织和 2 0例病人血浆的NPY浓度。结果 :不同类型垂体腺瘤组织中NPY含量有显著差别 ,侵袭性垂体腺瘤NPY含量高于非侵袭性垂体腺瘤。肿瘤组织与血浆NPY水平呈正相关。结论 :不同类型垂体腺瘤组织和血浆中NPY含量有显著差别 ,NPY水平可作为垂体腺瘤侵袭性的参考指标之一