Loss Of Feedback Inhibition Research Articles

Loss of feedback inhibition via D2 autoreceptors enhances acquisition of cocaine taking and reactivity to drug-paired cues.

A prominent aspect of drug addiction is the ability of drug-associated cues to elicit craving and facilitate relapse. Understanding the factors that regulate cue reactivity will be vital for improving treatment of addictive disorders. Low availability of dopamine (DA) D2 receptors (D2Rs) in the striatum is associated with high cocaine intake and compulsive use. However, the role of D2Rs of nonstriatal origin in cocaine seeking and taking behavior and cue reactivity is less understood and possibly underestimated. D2Rs expressed by midbrain DA neurons function as autoreceptors, exerting inhibitory feedback on DA synthesis and release. Here, we show that selective loss of D2 autoreceptors impairs the feedback inhibition of DA release and amplifies the effect of cocaine on DA transmission in the nucleus accumbens (NAc) in vitro. Mice lacking D2 autoreceptors acquire a cued-operant self-administration task for cocaine faster than littermate control mice but acquire similarly for a natural reward. Furthermore, although mice lacking D2 autoreceptors were able to extinguish self-administration behavior in the absence of cocaine and paired cues, they exhibited perseverative responding when cocaine-paired cues were present. This enhanced cue reactivity was selective for cocaine and was not seen during extinction of sucrose self-administration. We conclude that low levels of D2 autoreceptors enhance the salience of cocaine-paired cues and can contribute to the vulnerability for cocaine use and relapse.

Abstract IA22: Rationale and current status of development of PI3K/TOR pathway inhibitors

Abstract The PI3K/AKT pathway is the most frequently mutated pathway in breast cancer, with mutation and/or amplification of the genes encoding the PI3K catalytic subunits p110α (PIK3CA) and p110β (PIK3CB), the PI3K regulatory subunit p85α (PIK3R1), receptor tyrosine kinases (RTKs) such as HER2 (ERBB2) and FGFR1, the PI3K activator K-Ras, the PI3K effectors AKT1, AKT2, and PDK1, and loss of the lipid phosphatases PTEN and INPP4B. The three genes PIK3CA, PIK3CB, and PIK3CD encode the homologous p110α , p110β, and p110δ isozymes, respectively. Expression of p110δ; is largely restricted to immune and hematopoietic cells, whereas p110α and p110β are ubiquitously expressed. The p110α isozyme is essential for signaling and growth of tumors driven by PIK3CA mutations, RTKs, and/or mutant Ras, whereas p110β lies downstream of GPCRs and has been shown to mediate tumorigenesis in PTEN-deficient cells. PIK3CA mutations are the most common genetic alterations of this pathway, where ≥80% occur within the helical (E542K and E545K) and kinase (H1047R) domains of p110α. Such mutations confer increased catalytic activity through different mechanisms, but both induce characteristics of cellular transformation including growth factor- and anchorage-independent growth, and resistance to anoikis. Several drugs targeting multiple levels of the PI3K network (i.e., PI3K, AKT, mTOR) have been developed (Table 1). A number of ATP-mimetics that bind competitively and reversibly to the ATP-binding pocket of p110 are in early clinical development. These include the pan-PI3K inhibitors BKM120, XL-147, PX-866, PKI-587, and GDC-0941, the p110α-specific inhibitors BYL719, GDC-0032, and INK-1117, the p110δ-specific inhibitor CAL-101, and the dual PI3K/mTOR inhibitors BEZ235, BGT226, PF-4691502, GDC-0980, and XL-765. The pan-PI3K and p110α-specific inhibitors are equally potent against oncogenic p110α mutants. The rationale for the development of isozyme-specific antagonists is to allow higher doses of anti-p110α and anti-p110β drugs to be delivered without incurring side-effects caused by pan-PI3K inhibitors. Recently completed phase I trials with BKM120, BEZ235, and XL-147 showed that treatment partially inhibited PI3K as measured by levels of P-S6 and P-AKT in patients' skin or tumors, and 2-deoxy-2-[18F]fluoro-D-glucose (FDG) uptake measured by PET. Main toxicities were rash, hyperglycemia, diarrhea, fatigue and, mood alterations. Few clinical responses were observed in patients with and without detectable PI3K pathway mutations, although screening for genetic lesions in this pathway was not comprehensive. Both allosteric and ATP-competitive pan-inhibitors of the three isoforms of AKT are also being developed. AZD5363, GDC-0068, GSK2141795, and GSK690693 are ATP-competitive compounds which have shown antitumor activity in preclinical models and recently entered phase I trials. Allosteric inhibitors such as MK-2206 bind to the AKT PH domain and/or hinge region to promote an inactive conformation of the AKT protein that is unable to bind to the plasma membrane. MK-2206 inhibits AKT signaling in vivo, and suppresses growth of breast cancer xenografts harboring PIK3CA mutations or ERBB2 amplification. Phase I data showed that treatment with MK-2206 decreases levels of P-AKT, P-PRAS40, and P-GSK3β in tumor cells, peripheral blood mononuclear cells, and hair follicles. Another approach to block this pathway has been the development of ATP-competitive inhibitors of the mTOR kinase, which block both mTORC1 and mTORC2. Several dual TORC1/2 inhibitors have been identified, including INK128 (Intellikine), CC223 (Celgene), AZD2014 (AstraZeneca), and Palomid 529 (Paloma Pharmaceuticals). Dual PI3K/mTOR inhibitors have also been developed in the hope of overcoming the loss of feedback inhibition or PI3K activation observed with rapalogs. The mTORC1 pathway is one of the prominent negative feedback regulators of the PI3K pathway; inhibition of mTORC1 can release this feedback inhibition and activate the PI3K pathway19. BEZ235, a dual PI3K/mTOR inhibitor, showed higher anti-proliferative activity than rapamycin in a preclinical study trastuzumab-resistant and –sensitive human breast cancer cell lines. In breast cancer, dual PI3K/mTOR inhibitors are being combined with everolimus, endocrine therapies (exemestane and letrozole), chemotherapy (paclitaxel), and anti-HER2 therapy (trastuzumab). Citation Format: Carlos L. Arteaga. Rationale and current status of development of PI3K/TOR pathway inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA22.

TSH-Secreting Pituitary Macroadenoma in a Girl with Lingual Thyroid

Patients with long-standing hypothyroid are, in some cases, reported to develop pituitary gland hyperplasia due to loss of feedback inhibition of thyroxine in hypothalamus—the condition of which typically regresses after thyroxine replacement. Herein, a 15-year-old girl—with long-standing untreated lingual hypothyroid—presents with a pathologically proven TSH pituitary macroadenoma and bilateral large ovarian cysts. Although MR imaging may differentiate between hyperplasia and macroadenoma of the pituitary gland, pathological examination is still a cornerstone to correct diagnosis.

Thyroxine-induced expression of pyroglutamyl peptidase II and inhibition of TSH release precedes suppression of TRH mRNA and requires type 2 deiodinase

Suppression of TSH release from the hypothyroid thyrotrophs is one of the most rapid effects of 3,3',5'-triiodothyronine (T(3)) or thyroxine (T(4)). It is initiated within an hour, precedes the decrease in TSHβ mRNA inhibition and is blocked by inhibitors of mRNA or protein synthesis. TSH elevation in primary hypothyroidism requires both the loss of feedback inhibition by thyroid hormone in the thyrotrophs and the positive effects of TRH. Another event in this feedback regulation may be the thyroid hormone-mediated induction of the TRH-inactivating pyroglutamyl peptidase II (PPII) in the hypothalamic tanycytes. This study compared the chronology of the acute effects of T(3) or T(4) on TSH suppression, TRH mRNA in the hypothalamic paraventricular nucleus (PVN), and the induction of tanycyte PPII. In wild-type mice, T(3) or T(4) caused a 50% decrease in serum TSH in hypothyroid mice by 5 h. There was no change in TRH mRNA in PVN over this interval, but there was a significant increase in PPII mRNA in the tanycytes. In mice with genetic inactivation of the type 2 iodothyronine deiodinase, T(3) decreased serum TSH and increased PPII mRNA levels, while T(4)-treatment was ineffective. We conclude that the rapid suppression of TSH in the hypothyroid mouse by T(3) occurs prior to a decrease in TRH mRNA though TRH inactivation may be occurring in the median eminence through the rapid induction of tanycyte PPII. The effect of T(4), but not T(3), requires the type 2 iodothyronine deiodinase.

Down‐regulation of serum gonadotropins is as effective as estrogen replacement at improving menopause‐associated cognitive deficits

Declining levels of estrogen in women result in increases in gonadotropins such as luteinizing hormone (LH) through loss of feedback inhibition. LH, like estrogen, is modulated by hormone replacement therapy. However, the role of post-menopausal gonadotropin increases on cognition has not been evaluated. Here, we demonstrate that the down-regulation of ovariectomy-driven LH elevations using the gonadotropin releasing hormone super-analogue, leuprolide acetate, improves cognitive function in the Morris water maze and Y-maze tests in the absence of E2. Furthermore, our data suggest that these effects are independent of the modulation of estrogen receptors alpha and beta, or activation of CYP19 and StAR, associated with the production of endogenous E2. Importantly, pathways associated with improved cognition such as CaMKII and GluR1-Ser831 are up-regulated by leuprolide treatment but not by chronic long-term E2 replacement suggesting independent cognition-modulating properties. Our findings suggest that down-regulation of gonadotropins is as effective as E2 in modulating cognition but likely acts through different molecular mechanisms. These findings provide a potential novel protective strategy to treat menopause/age-related cognitive decline and/or prevent the development of AD.

Zac1 functions through TGFβIIto negatively regulate cell number in the developing retina

BackgroundOrgans are programmed to acquire a particular size during development, but the regulatory mechanisms that dictate when dividing progenitor cells should permanently exit the cell cycle and stop producing additional daughter cells are poorly understood. In differentiated tissues, tumor suppressor genes maintain a constant cell number and intact tissue architecture by controlling proliferation, apoptosis and cell dispersal. Here we report a similar role for two tumor suppressor genes, the Zac1 zinc finger transcription factor and that encoding the cytokine TGFβII, in the developing retina.ResultsUsing loss and gain-of-function approaches, we show that Zac1 is an essential negative regulator of retinal size. Zac1 mutants develop hypercellular retinae due to increased progenitor cell proliferation and reduced apoptosis at late developmental stages. Consequently, supernumerary rod photoreceptors and amacrine cells are generated, the latter of which form an ectopic cellular layer, while other retinal cells are present in their normal number and location. Strikingly, Zac1 functions as a direct negative regulator of a rod fate, while acting cell non-autonomously to modulate amacrine cell number. We implicate TGFβII, another tumor suppressor and cytokine, as a Zac1-dependent amacrine cell negative feedback signal. TGFβII and phospho-Smad2/3, its downstream effector, are expressed at reduced levels in Zac1 mutant retinae, and exogenous TGFβII relieves the mutant amacrine cell phenotype. Moreover, treatment of wild-type retinae with a soluble TGFβ inhibitor and TGFβ receptor II (TGFβRII) conditional mutants generate excess amacrine cells, phenocopying the Zac1 mutant phenotype.ConclusionWe show here that Zac1 has an essential role in cell number control during retinal development, akin to its role in tumor surveillance in mature tissues. Furthermore, we demonstrate that Zac1 employs a novel cell non-autonomous strategy to regulate amacrine cell number, acting in cooperation with a second tumor suppressor gene, TGFβII, through a negative feedback pathway. This raises the intriguing possibility that tumorigenicity may also be associated with the loss of feedback inhibition in mature tissues.

Differential control of adrenal and sympathetic catecholamine release by alpha 2-adrenoceptor subtypes.

In the adrenergic system, release of the neurotransmitter norepinephrine from sympathetic nerves is regulated by presynaptic inhibitory alpha2-adrenoceptors, but it is unknown whether release of epinephrine from the adrenal gland is controlled by a similar short feedback loop. Using gene-targeted mice we demonstrate that two distinct subtypes of alpha2-adrenoceptors control release of catecholamines from sympathetic nerves (alpha 2A) and from the adrenal medulla (alpha 2C). In isolated mouse chromaffin cells, alpha2-receptor activation inhibited the electrically stimulated increase in cell capacitance (a correlate of exocytosis), voltage-activated Ca2+ current, as well as secretion of epinephrine and norepinephrine. The inhibitory effects of alpha2-agonists on cell capacitance, voltage-activated Ca2+ currents, and on catecholamine secretion were completely abolished in chromaffin cells isolated from alpha 2C-receptor-deficient mice. In vivo, deletion of sympathetic or adrenal feedback control led to increased plasma and urine norepinephrine (alpha 2A-knockout) and epinephrine levels (alpha 2C-knockout), respectively. Loss of feedback inhibition was compensated by increased tyrosine hydroxylase activity, as detected by elevated tissue dihydroxyphenylalanine levels. Thus, receptor subtype diversity in the adrenergic system has emerged to selectively control sympathetic and adrenal catecholamine secretion via distinct alpha2-adrenoceptor subtypes. Short-loop feedback inhibition of epinephrine release from the adrenal gland may represent a novel therapeutic target for diseases that arise from enhanced adrenergic stimulation.

Endogenous glucocorticoids restrain catecholamine synthesis and release at rest and during immobilization stress in rats

Cardiovascular and metabolic adjustments during stress involve participation of the sympatho-adrenal and hypothalamic-pituitary-adrenocortical systems, which interact at several levels. The present study investigated the effects of removal of endogenous glucocorticoids on indices of norepinephrine release and metabolism and of catecholamine biosynthesis in sympathetic nerves, at baseline and in response to immobilization stress (IMMO) in rats. Plasma levels of the catecholamine precursor dihydroxyphenylalanine, norepinephrine (NE), the NE metabolites dihydroxyphenylglycol and methoxyhydroxyphenylglycol, dopamine, and the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid were measured in adrenalectomized, adrenal-medullectomized, or sham-operated conscious rats, with or without glucocorticoid treatment (25 mg/kg.24 h cortisol for 7 days by osmotic minipumps), at rest and after 5, 20, 60, and 120 min of IMMO. Adrenalectomy eliminated plasma levels of epinephrine and corticosterone and augmented IMMO-induced increments in levels of NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, dihydroxyphenylalanine, dihydroxyphenylacetic acid, and homovanillic acid. Adrenal-medullectomy reduced plasma epinephrine, but not corticosterone, to undetectable levels at baseline and did not augment stress-induced responses of any of the measured compounds. Cortisol administration (plasma levels approximately 600 pmol/ml) reversed the augmentation of catecholaminergic responses in adrenalectomized rats. The results indicate that adrenalectomy stimulates several aspects of sympatho-neural function, including stress-induced increments in NE release, reuptake, metabolism, and turnover and in catecholamine biosynthesis. This augmentation was absent in adreno-medullectomized rats, indicating that the loss of adrenomedullary hormones after adrenalectomy does not appear to contribute to the augmentation. Since cortisol treatment reversed or prevented this augmentation, loss of feedback inhibition by endogenous glucocorticoids appears to be the basis of the enhanced responses. Thus, the results suggest that endogenous glucocorticoids restrain responses of catecholamine turnover, synthesis, release, reuptake, and metabolism in sympathetic nerves in this stress model.

Endogenous glucocorticoids restrain catecholamine synthesis and release at rest and during immobilization stress in rats.

Cardiovascular and metabolic adjustments during stress involve participation of the sympatho-adrenal and hypothalamic-pituitary-adrenocortical systems, which interact at several levels. The present study investigated the effects of removal of endogenous glucocorticoids on indices of norepinephrine release and metabolism and of catecholamine biosynthesis in sympathetic nerves, at baseline and in response to immobilization stress (IMMO) in rats. Plasma levels of the catecholamine precursor dihydroxyphenylalanine, norepinephrine (NE), the NE metabolites dihydroxyphenylglycol and methoxyhydroxyphenylglycol, dopamine, and the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid were measured in adrenalectomized, adrenal-medullectomized, or sham-operated conscious rats, with or without glucocorticoid treatment (25 mg/kg.24 h cortisol for 7 days by osmotic minipumps), at rest and after 5, 20, 60, and 120 min of IMMO. Adrenalectomy eliminated plasma levels of epinephrine and corticosterone and augmented IMMO-induced increments in levels of NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, dihydroxyphenylalanine, dihydroxyphenylacetic acid, and homovanillic acid. Adrenal-medullectomy reduced plasma epinephrine, but not corticosterone, to undetectable levels at baseline and did not augment stress-induced responses of any of the measured compounds. Cortisol administration (plasma levels approximately 600 pmol/ml) reversed the augmentation of catecholaminergic responses in adrenalectomized rats. The results indicate that adrenalectomy stimulates several aspects of sympatho-neural function, including stress-induced increments in NE release, reuptake, metabolism, and turnover and in catecholamine biosynthesis. This augmentation was absent in adreno-medullectomized rats, indicating that the loss of adrenomedullary hormones after adrenalectomy does not appear to contribute to the augmentation. Since cortisol treatment reversed or prevented this augmentation, loss of feedback inhibition by endogenous glucocorticoids appears to be the basis of the enhanced responses. Thus, the results suggest that endogenous glucocorticoids restrain responses of catecholamine turnover, synthesis, release, reuptake, and metabolism in sympathetic nerves in this stress model.

TyrR, a regulatory gene of tyrosine biosynthesis in Salmonella typhimurium.

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which tyrosine pathway enzymes were not repressed by l-tyrosine. The mutants produced elevated levels of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase, and these enzymes as well as transaminase A were not repressed by high concentrations of tyrosine. Genetic analysis revealed that a mutation in a gene designated tyrR was responsible for the constitutivity of the tyrosine pathway enzymes in strains SG1, SG7, and SG9, and that tyrR was linked to pyrF. In strain SG1 a mutation had also occurred in aroF, the structural gene for DAHP synthetase (tyr), resulting in loss of sensitivity of this enzyme to end-product inhibition. There appeared to be no relationship between loss of feedback inhibition and loss of end-product repression, since derivative strains of SG1 that carried only the tyrR mutation behaved like the singly mutated tyrR strains, SG7 and SG9, in showing high constitutive levels of tyrosine-specific enzymes that were not repressed by tyrosine.

Kinetics and regulation of the salt-dependent aspartate transcarbamylase of Halobacterium cutirubrum.

Properties of the aspartate transcarbamylase of the extremly halophilic bacterium Halobacterium cutirubrum, an enzyme that needs high salt concentrations for activity and regulation, were studied in cell-free extracts. The enzyme was stable on prolonged incubation at 4 C in concentrated extracts (50 mg of protein per ml) but not in diluted extracts. Mg(2+) ions and beta-mercaptoethanol stabilized enzyme activity. At salt concentrations below the maximum for activity (3.5 m), the enzyme was rapidly inactivated. Carbamyl phosphate stabilized the enzyme under these conditions; aspartate had a smaller effect. The enzyme was most stable at 0 C; raising or lowering the temperature from this point increased the rate of inactivation. On exposure to lowered salt concentrations, enzyme activity was more sensitive than feedback inhibition. Hyperbolic substrate saturation curves were found for carbamyl phosphate. The K(m) obtained varied with the salt concentration used. With aspartate, sigmoidal curves were found when extracts were assayed immediately after preparation, but hyperbolic curves were obtained with extracts allowed to stand 1 to 2 hr. The presence of cytidine triphosphate (CTP) decreased the V(max) but did not change the K(m); this is thus a V-type enzyme. Low concentrations of succinate activated the enzyme, in the presence and absence of CTP; higher concentrations did not affect its activity. CTP increased the activation energy of the enzyme in 3.5 m salt but decreased it in 2.0 m salt. At both salt concentrations, the sensitivity of the enzyme to feedback inhibition diminished with increasing temperatures. Gel chromatography suggested that the enzyme in crude extracts had a molecular weight of 160,000. Precipitating the enzyme with polyethylene glycol decreased the molecular weight to 34,000, and this activity was no longer sensitive to CTP. The presence of either substrate of the enzyme during polyethylene glycol treatment prevented dissociation of the enzyme and loss of feedback inhibition. Thus, as with other aspartate transcarbamylases, association of subunits seems to be required for regulation of activity by end product.

The combined effects of temperature and dilution on the activity and feedback inhibition of yeast aspartate transcarbamylase

Dilution of a partially purified preparation of yeast aspartate transcarbamylase caused only a small loss of feedback inhibition and no change in specific activity when the assay was carried out at low temperature (0–10 °C). As the temperature of assay was increased, there was in the case of the dilute preparation a progressive loss of feedback inhibition coupled with a sharp increase in specific activity, reaching a level 400–500% that of the concentrated preparations. The data suggest that this is due to a dissociation into subunits possessing high aspartate transcarbamylase activity but lacking the regulatory site.