The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
BACKGROUND - We examined the activation pattern of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and its dependence on D1 versus D2 dopamine receptors in hemiparkinsonian rats treated with 3,4-dihydroxyphenyl-L-alanine (L-DOPA).
METHODS - 6-Hydroxydopamine-lesioned rats were treated acutely or chronically with L-DOPA in combination with antagonists for D1 or D2 receptors. Development of dyskinesia was monitored in animals receiving chronic drug treatment. Phosphorylation of ERK1/2, mitogen- and stress-activated protein kinase-1 (MSK-1), and the levels of FosB/DeltaFosB expression were examined immunohistochemically.
RESULTS - L-DOPA treatment caused phosphorylation of ERK1/2 in the dopamine-denervated striatum after acute and chronic administration. Similar levels were observed in matrix and striosomes, and in enkephalin-positive and dynorphin-positive neurons. The severity of dyskinesia was positively correlated with phospho-ERK1/2 levels. Phosphorylation of ERK1/2 and MSK-1 was dose-dependently blocked by SCH23390, but not by raclopride. SCH23390 also inhibited the development of dyskinesia and the induction of FosB/DeltaFosB.
CONCLUSIONS - L-DOPA produces pronounced activation of ERK1/2 signaling in the dopamine-denervated striatum through a D1-receptor-dependent mechanism. This effect is associated with the development of dyskinesia. Phosphorylated ERK1/2 is localized to both dynorphinergic and enkephalinergic striatal neurons, suggesting a general role of ERK1/2 as a plasticity molecule during L-DOPA treatment.
Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.
Enkephalin modulates striatal function, thereby affecting motor performance and addictive behaviors. The proenkephalin gene is also used as a model to study cyclic AMP-mediated gene expression in striatal neurons. The second messenger pathway leading to proenkephalin expression demonstrates how cyclic AMP pathways are synchronized with depolarization. We show that cyclic AMP-mediated regulation of the proenkephalin gene is dependent on the activity of L-type Ca2+ channels. Inhibition of L-type Ca2+ channels blocks forskolin-mediated induction of proenkephalin. The Ca2+-activated kinase, Ca2+/calmodulin kinase, as well as the cyclic AMP-activated kinase, protein kinase A (PKA), are both necessary for the induction of the proenkephalin promoter. Similarly, both kinases are needed for the L-type Ca2+ channel-mediated induction of proenkephalin. This synchronization of second messenger pathways provides a coincidence mechanism that gates proenkephalin synthesis in striatal neurons, ensuring that levels are increased only in the presence of activated PKA and depolarization.
The cAMP response element-binding protein (CREB) is believed to play a pivotal role in dopamine (DA) receptor-mediated nuclear signaling and neuroplasticity. Here we demonstrate that the significance of CREB for gene expression depends on the experimental paradigm. We compared the role of CREB in two different but related models: l-DOPA administration to unilaterally 6-hydroxydopamine lesioned rats, and cocaine administration to neurologically intact animals. Antisense technology was used to produce a local knockdown of CREB in the lateral caudate-putamen, a region that mediates the dyskinetic or stereotypic manifestations associated with l-DOPA or cocaine treatment, respectively. In intact rats, CREB antisense reduced both basal and cocaine-induced expression of c-Fos, FosB/DeltaFosB, and prodynorphin mRNA. In the DA-denervated striatum, CREB was not required for l-DOPA to induce these gene products, nor did CREB contribute considerably to DNA binding activity at cAMP responsive elements (CREs) and CRE-like enhancers. DeltaFosB-related proteins and JunD were the main contributors to both CRE and AP-1 DNA-protein complexes in l-DOPA-treated animals. In behavioral studies, intrastriatal CREB knockdown caused enhanced activity scores in intact control animals and exacerbated the dyskinetic effects of acute l-DOPA treatment in 6-OHDA-lesioned animals. These data demonstrate that CREB is not required for the development of l-DOPA-induced dyskinesia in hemiparkinsonian rats. Moreover, our results reveal an unexpected alteration of nuclear signaling mechanisms in the parkinsonian striatum treated with l-DOPA, where AP-1 transcription factors appear to supersede CREB in the activation of CRE-containing genes.
The in vivo metabolism of peptide E was studied in the anesthetized rat using a combination of microdialysis sampling, solid-phase preconcentration capillary electrophoresis and imaging matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). The metabolic profile of peptides identified by MALDI/MS showed that the primary enzymatic activity for degradation of peptide E was due to carboxypeptidases and, to a lesser extent, aminopeptidases and some trypsin-like endopeptidases. Over 75 metabolic fragments were detected from the action of these enzymes in vivo.
17beta-Estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic (beta-endorphin) neurons. In the current study, we used intracellular recordings from guinea pig hypothalamic slices to characterize the receptor and intracellular effector system mediating the rapid effects of E2. E2 acted stereospecifically with physiologically relevant concentration dependence (EC50 = 8 nM) to cause a 4-fold reduction in the potency of a mu-opioid agonist to activate an inwardly rectifying K+ conductance. Using Schild analysis to estimate the affinity of the mu-opioid receptor for an antagonist (naloxone), we found that estrogen did not compete for the mu-opioid receptor or alter the affinity of the mu receptor. Both the nonsteroidal estrogen diethylstilbestrol and the "pure" antiestrogen ICI 164,384 blocked the actions of E2, the latter with a subnanomolar affinity. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a nongenomic mechanism of action by E2. The actions of E2 were mimicked by the protein kinase A (PKA) activators forskolin and cAMP, Sp-isomer triethylammonium salt. Furthermore, the selective PKA antagonists cAMP, Rp-isomer triethylammonium salt and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. Thus, estrogen binds to a specific receptor that activates PKA to rapidly uncouple the mu-opioid receptor from its K+ channel. Because we have previously shown that gamma-aminobutyric acidB receptors are also uncoupled by estrogen, this mechanism of action has the potential to alter synaptic transmission via G protein-coupled receptors throughout the brain.
The present study examined the potential for cross-tolerance development between mu-opioid and gamma-aminobutyric acidB receptor agonists, in hypothalamic arcuate neurons, resulting from chronic morphine treatment. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized female guinea pigs. The mu-opioid receptor agonist D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin and the gamma-aminobutyric acidB receptor agonist baclofen produced dose-dependent membrane hyperpolarizations of arcuate neurons. The reversal potential for both agonist-induced hyperpolarizations was near -95 mV, indicative of the activation of an underlying K+ conductance. Coadministration of maximally effective concentrations of D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin and baclofen produced a response that was not additive, indicating a convergence onto a common K+ channel. In arcuate neurons, including a subset that was immunopositive for tyrosine hydroxylase, chronic morphine treatment for 4 to 7 days produced a 3.2-fold reduction in the potency, with no change in the efficacy, of D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin. In contrast, it affected neither the potency nor the efficacy of baclofen. Therefore, chronic morphine exposure does not produce cross-tolerance between mu-opioid and gamma-aminobutyric acidB receptor agonists in A12 dopamine neurons, suggesting that convergence upon a common effector is not a sufficient criterion for the development of cross-tolerance between receptor systems.
GABA is a predominant neurotransmitter in the hypothalamus and an important regulator of hypothalamic function. To elucidate the cellular basis for GABAergic action in this region, we used intracellular recordings from identified hypothalamic neurons. Ninety-three percent of the mediobasal hypothalamic neurons responded to GABAB receptor stimulation, and the presence of bicuculline-sensitive synaptic potentials indicated a tonic, GABAA receptor-mediated input. Stimulation of GABAB receptors hyperpolarized these cells by activating an inwardly rectifying potassium conductance. We characterized GABAB responses by generating concentration-response curves to the GABAB agonist baclofen. There was heterogeneity in the responses to baclofen, with one third of the cells having low baclofen potency (EC50 = 5.0 microM). Two thirds of the neurons had a 4-fold higher potency (EC50 = 1.2 microM), larger somas and a more lateral distribution. Previous work has shown that hypothalamic GABAB and mu-opioid receptors open the same K+ channels and that the response to mu-opioid agonists is rapidly attenuated by 17 beta-estradiol (E2). In order to test the hypothesis that the coupling of GABAB receptors to K+ channels is also altered, baclofen concentration-response curves were generated before and after an E2 challenge (100 nM, 20 min). Consistent with our hypothesis, the potency of baclofen was decreased nearly 4-fold in a subset of the cells that had a high potency response to baclofen. Furthermore, decreased baclofen potency only occurred in those cells in which E2 also altered the mu-opioid responses. Therefore, our findings suggest that a discrete subpopulation of hypothalamic neurons is sensitive to estrogen actions to alter inhibitory transmission. We propose that the alteration of GABAB and mu-opioid input is consistent with estrogen's rapid inhibition of the reproductive axis.
The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors. Here, we report that Leu- and Met-enkephalin and their des-tyrosyl derivatives potently and selectively inhibited high affinity L-proline uptake in rat hippocampal synaptosomes and in PROT-transfected HeLa cells. High concentrations of the opiate receptor antagonist naltrexone did not block the inhibitory actions of these peptides, arguing against an involvement of opioid receptors. Des-tyrosyl-Leu-enkephalin elevated the apparent K(m) of L-proline transport in transfected HeLa cells without altering the V(max). PROT-transfected HeLa cells did not accumulate [3H]Leu-enkephalin above background levels, demonstrating that enkephalins are not substrates for PROT. These findings indicate that enkephalins competitively inhibit mammalian brain PROT through a direct interaction with the transporter protein at or near the L-proline binding site. The high potency and specificity of des-tyrosyl-Leu-enkephalin make this compound a useful tool for elucidating the structure-function properties and physiological role(s) of PROT.
Modulation of voltage-gated Ca(2+) channel current (I(Ca)) regulates secretion of catecholamines from adrenal chromaffin cells. Previous work demonstrated that I(Ca) can be augmented by phosphorylation to increase secretion or that inhibition of I(Ca) results in diminished catecholamine secretion. In the current manuscript, we show that stimulation of chromaffin cells results in the release of an "endogenous inhibitor" that suppresses I(Ca). The inhibition is due to the secretion of ATP, which is stored at high concentrations in secretory granules and is coreleased with catecholamines upon stimulation. The ATP exerts its actions through P(2 gamma) purinoceptors and inhibits both N- and P/Q-type Ca (2+) channels in a voltage-dependent manner but with different efficacies. Overall, we have identified and characterized a negative feedback pathway that may serve as an important regulatory mechanism for catecholamine secretion in chromaffin cells.