GLC-ASPET Speaker Abstracts 2011
PKC regulation of cys-loop receptors in alcohol and nicotine addiction
Gallo Center, University of California at San Francisco
Alcohol and nicotine addiction are major public health problems with an estimated combined annual cost of over $400 billion dollars in the US alone. Given the magnitude of the problem, it is surprising that there are few pharmacotherapic agents available to treat patients with these disorders. My laboratory is focused on identifying biochemical mechanisms involved in drug action that control self-administration of these drugs in animals. This lecture will focus on studies of GABAA and nicotinic cholinergic receptors, members of the cysloop superfamily of receptor-operated channels, and their regulation by members of the protein kinase C (PKC) family of serine-threonine kinases. I will discuss our work with PKC epsilon and PKC delta in the regulation of GABAA receptor trafficking and sensitivity to alcohol, and recent studies on PKC epsilon regulation of alpha 6-containing nicotinic receptors in responses to nicotine. These studies highlight PKC epsilon as a potential drug target for the treatment of co-morbid alcohol and nicotine addiction.
Why is PDE10A inhibition predicted to be antipsychotic?
Christopher J Schmidt, Ph.D.
Neuroscience Research Unit
Groton, CT 06340
The psychosis associated with schizophrenia is the result of excessive dopaminergic activity within the striatum, the primary nucleus of a subcortical circuit involved in the regulation of cortically initiated responses to environmental cues. It is at the level of the striatum that cortically selected actions are inhibited or reinforced by ascending dopaminergic inputs from the midbrain. Environmental cues predictive of positive or rewarding outcomes are known to increase striatal dopamine release thereby enhancing the activity of those corticostriatal connections selecting behaviors in pursuit of that outcome while simultaneously inhibiting conflicting behaviors. Cues predicting negative outcome or the loss of a reward result in reduced dopaminergic signaling and favor the selection of avoidance behaviors. The elevated striatal dopamine release
found in schizophrenia explains why inappropriate behaviors are not inhibited but rather reinforced and maintained despite their potentially negative consequences. Antipsychotic agents block the action of
dopamine at D2 receptors to allow cortical drive of the striatal neurons involved in the suppression of unwanted behaviors. PDE10A is a dual substrate phosphodiesterase expressed at very high levels in the
medium spiny neurons (MSNs) of the striatum where it regulates the interaction of glutamatergic and dopaminergic signals by controlling intracellular levels of cAMP and cGMP. Studies conducted in our laboratories confirm that the inhibition of PDE10A can directly oppose the actions of dopamine at D2 receptor on MSNs to enhance their response to cortical drive. We demonstrate that PDE10A inhibition reproduces a majority of the therapeutically relevant biochemical effects of D2 receptor antagonists and is therefore predicted to exhibit antipsychotic activity in the clinic.
MAP kinase phosphatases as therapeutic targets for human diseases
Anton M. Bennett
Yale University School of Medicine, Department of Pharmacology and Program in Integrative Cell Signaling and
Neurobiology of Metabolism, 333 Cedar Street, New Haven, CT 06520
The mitogen-activated protein kinases (MAPKs) have been implicated in a number of pathologies including cancer, obesity, type 2 diabetes, cardiovascular disease, inflammatory and neurological diseases. These observations suggest that the MAPKs, and those signaling components that regulate them, can serve potentially as therapeutic targets to combat these complex diseases. The MAPK phosphatases (MKPs) mediate the dephosphorylation and inactivation of the MAPKs in response to growth factors, stress and electrophysiological stimuli. Therefore, the actions of the MKPs play an important role in the maintenance of physiological MAPK-mediated signaling. Although much work on the MKPs in recent years has demonstrated that this family of enzymes plays essential roles in regulating MAPK-dependent processes a role for the MKPs in disease progression is less clear. We have been investigating the actions of the MKPs in metabolic, inflammatory and musculoskeletal diseases. This work has suggested that the MKPs may play an important role in the progression of obesity and type 2 diabetes, in addition to musculoskeletal disorders such as the muscular dystrophies. We will highlight recent work from this laboratory to support the notion that the MKPs potentially represent meaningful therapeutic targets for the treatment of certain human diseases.
Akt’s functions in cancer and diabetes: Implications for therapy
Department of Biochemistry and Molecular Genetics
The University of Illinois at Chicago
The serine/threonine kinase Akt, also known as protein kinase B, is an evolutionarily conserved downstream effector of Insulin/IGF1 receptor and PI3K. In recent years Akt attracted a substantial attention largely because of its frequent hyperactivation in human cancers. However, the most conserved function of Akt is in the regulation of metabolism, and glucose homeostasis at cellular and organismal levels. Using genetics in mice we delineated the in vivo functions of the three Akt isoforms. We showed that Akt is required for oncogenic transformation, and that the deficiency of Akt1 in mice is sufficient to render them resistant to cancer. The function of Akt in cell proliferation and oncogenic transformation is coupled to its role in cellular energy metabolism. Finally, we delineated the functions of Akt isoforms in glucose homeostasis and in diabetes. We analyzed the contributions of the 3 Akt isoforms to diabetes. The diabetic phenotype in Akt-deficient mice is associated with reduced circulating leptin levels, and restoring leptin levels restores normal glucose homeostasis in diabetic Akt-deficient mice.
GLC-ASPET Young Investigator Symposium 2011
Adaptive variation regulates the expression of the human SGK1 gene in response to glucocorticoids
Francesca Luca, PhD
Department of Human Genetics, University of Chicago, Chicago, IL 60637
Glucocorticoids (GCs) are steroid hormones that mediate the response to stress. GCs modulate a large number of biological processes (e.g.: inflammation) and are widely used as pharmacological agents in the treatment of inflammatory diseases, and cancer (e.g. leukemia). GC action is largely mediated by the interaction with the GC receptor (GR), which activates a transcriptional cascade by regulating gene expression of direct targets. The Serum and Glucocorticoid-regulated Kinase1 (SGK1) gene is a target of the GR and is central to the stress response in many human tissues. Because environmental stress varies across habitats, we hypothesized that natural selection shaped the geographic distribution of genetic variants regulating the level of SGK1 expression following GR activation. By combining population genetics and molecular biology methods, we identified a variant with marked allele frequency differences between populations of African and European ancestry and with a strong correlation between allele frequency and latitude in worldwide population samples. This SNP is located in a GR-binding region upstream of SGK1 and within a predicted binding site for Oct1, a transcription factor known to interact with the GR. Using ChIP assays, we showed that both GR and Oct1 bind to this region and that the ancestral allele binds the GR-Oct1 complex more efficiently than the derived allele. Finally, using a reporter gene assay, we demonstrated that the ancestral allele is associated with increased GC-dependent gene expression relative to the derived allele. Our results suggest a novel paradigm in which hormonal responsiveness is modulated by sequence variation in the regulatory regions of nuclear receptor target genes. Identifying such functional variants may shed light on the mechanisms underlying inter-individual variation in response to environmental stressors and to hormonal therapy, as well as in the susceptibility to hormone dependent diseases.
SSRI antidepressants potentiate psychostimulant (Ritalin)-induced gene regulation: risk for enhanced
Vincent Van Waes, PhD
Department of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science /
The Chicago Medical School, North Chicago, IL 60064
Current Address: Laboratoire de Neurosciences Integratives et Cliniques, Université de Franche-Compté –
Besancon, France ; Email: firstname.lastname@example.org
The use of psychostimulants such as methylphenidate (Ritalin), both in the treatment of attention-deficit hyperactivity disorder and as “cognitive enhancers” in the healthy, has increased considerably over the past decades. Methylphenidate, like cocaine, acts by blocking the reuptake of dopamine and norepinephrine. However, unlike cocaine, methylphenidate does not affect serotonin. Studies show that serotonin contributes to cell-signaling effects of cocaine. This may explain why methylphenidate mimics some but not all of the molecular effects of cocaine. We investigated whether serotonin-enhancing medications such as selective serotonin reuptake inhibitors (SSRIs; fluoxetine, citalopram) modify molecular and behavioral effects of methylphenidate. We assessed molecules known to be upregulated by cocaine and other addictive drugs: the transcription factors zif 268 and c-fos, and the neuropetides substance P and dynorphin in the striatum. Our results show that fluoxetine and citalopram robustly potentiate methylphenidate-induced gene regulation throughout the striatum. This potentiation was most pronounced in the sensorimotor striatum. Significant but smaller effects were also seen in subregions of the nucleus accumbens. At the behavioral level, SSRIs did not affect methylphenidate-induced locomotion, but potentiated stereotypies. Further studies showed that methylphenidate+fluoxetine treatment triggers robust reinstatement of cocaine seeking behavior in an animal model of relapse. Together, these findings show that the molecular effects of the methylphenidate+SSRI combination mimic cocaine effects more closely than those of methylphenidate alone, and suggest that SSRI antidepressants potentiate the addiction liability of methylphenidate. This is of concern as methylphenidate+SSRI concomitant therapies are indicated in several neuropsychiatric disorders, and coexposure may also occur with cognitive enhancer use by patients on SSRIs.
Integrin Crosstalk in Endothelial Cells Is Regulated by Protein Kinase A and Protein Phosphatase 1
Annette M Gonzalez, PhD
Department of Pathology, Northwestern University, Chicago, IL 60611
My research interest is in examining integrin-laminin interactions in endothelial cells and how this impacts adhesion and blood vessel development in tumors. Angiogenesis is a complex process that not only depends on growth factors and their receptors but is also influenced by integrin-extracellular matrix interactions. We have identified the alpha 4 laminin, a component of the basement membrane of blood vessels as a ligand for αvβ3 integrin in endothelial cells. We have mapped the binding site of αvβ3 integrin to the carboxy-terminal, globular domain of the alpha 4 laminin subunit and have shown that the alpha 4 laminin subunit plays a key role in angiogenesis in vivo. Moreover, we have uncovered a novel mechanism by which β1 integrin can negatively cross-talk with αvβ3 integrin and regulate its function. We have shown that crosstalk between β1 integrin and αvβ3 integrin involves protein kinase A and components of the inhibitor-1 pathway. Indeed, a phosphatase and protein kinase A appear to act antagonistically in endothelial cells to modulate αvβ3 integrinmediated cell adhesion to alpha 4 laminin, a process that is likely to be an important regulator of vasculogenesis, angiogenesis and blood vessel homeostasis. We have extended these studies in order to gain a better understanding of how integrins function together to regulate cellular processes including adhesion, migration and proliferation. Our results implicate β1 integrin as a master regulator of endothelial cell behavior and indicate that it exerts its functions, in part, by modulating the activity of protein kinase A.
GLC-ASPET Abstracts 2011: Faculty
Involvement of α2-adrenoceptors and imidazoline receptors in agmatine and BMS182874 induced changes in opioid
analgesia and hypothermia
Shaifali Bhalla, Shridhar V. Andurkar, Anil Gulati
Pharmaceutical Sciences, Chicago College of Pharmacy, Midwestern University, Downers Grove, IL 60515
Background and Objectives: Endothelin-A (ETA) receptor antagonist, BMS182874, potentiates opioid analgesia and prevents analgesic tolerance without affecting body temperature. Clonidine (imidazoline/α2-adrenoceptor agonist) enhances opioid analgesia via α2-adrenoceptors and possibly imidazoline receptors without affecting opioid hyperthermia in rats. An endogenous clonidine-like substance, agmatine also enhances morphine analgesia and blocks morphine hyperthermia in rats. However, analgesic interaction of agmatine and oxycodone, and agmatine’s effect on opioid hypothermia in mice are unknown. Objectives were to determine: 1) effect of agmatine on morphine and oxycodone analgesia and hypothermia with or without BMS182874; 2) involvement of ETA-receptors, α2- adrenoceptors, imidazoline receptors in the effect of agmatine and BMS182874 on opioid actions. Methods: Analgesia was determined by tail-flick latency method of D'Amour and Smith in male Swiss Webster mice. Body temperature was determined using Cole-Palmer colonic thermometer. Parameters were measured for 360min and expressed as Mean±S.E.M. N=6 per group. Results: Both agmatine and BMS182874, significantly enhance morphine and oxycodone analgesia. Combination of agmatine and BMS182874 did not further affect morphine (P=0.2536) or oxycodone (P=0.0883). Agmatine-induced increase in morphine and oxycodone analgesia was blocked by imidazoline/α2- adrenoceptor antagonist idazoxan (P=0.0188; P<0.0001, respectively). Idazoxan also blocked BMS182874-induced