PubMed Health⌕ Search

Biomedical subjects

Lakshmi A Devi

Publications and source records attributed to Lakshmi A Devi.

17 recordsLinked to original sources
PubMed2005

Serotonin receptor activation leads to neurite outgrowth and neuronal survival.

Serotonin 5-HT1 receptors are implicated in anxiety and depression. These receptors belong to the family A of G-protein-coupled receptors and couple to inhibitory G-proteins. Recent studies show that chronic activation of 5-HT1A receptors leads to proliferation of hippocampal neurons suggesting that neurogenesis contributes to the effects of antidepressants. However, the molecular mechanisms and pathways involved are not understood. We used Neuro 2A cells transfected with 5-HT1A receptors and SK-N-SH cells endogenously expressing the receptor to examine the effect of receptor activation on neuronal survival and neurite outgrowth. We find that receptor activation leads to increased neurite outgrowth that can be blocked by the receptor selective antagonist and by treatment with pertussis toxin or lactacystin implicating inhibitory G-proteins and proteasomal degradation in this process. Interestingly, the small G-protein Rap and the transcription factor STAT-3 are also involved since reducing the levels of Rap1 (using small interfering RNA) or STAT-3 (using dominant negative STAT3) significantly blocks 5-HT1A-receptor-mediated neurite outgrowth. The observed increase in the phosphorylation of Src and STAT-3, at sites leading to their activation, further supports a crucial role for these proteins in neurite outgrowth. We also find that prolonged activation of endogenous 5-HT1A receptors leads to increased cell survival even under starving conditions; this is completely blocked by co-treatment with the antagonist. Taken together, these findings indicate that activation of the 5-HT1A receptor leads to a number of neurotropic events by activating a series of signal transduction molecules leading to long-term changes required for neurogenesis.

Animals↗
PubMed2005

The G alpha(o/i)-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3.

The study of the signaling pathways regulating neurite outgrowth in culture is important because of their potential role in neuronal differentiation in vivo. We have previously shown that the G alpha(o/i)-coupled CB1 cannabinoid receptor (CB1R) activates Rap1 to induce neurite outgrowth. G alpha(o/i) also activates the Src-Stat3 pathway. Here, we studied the relationship between the G alpha(o/i)-Rap1 and Src-Stat3 pathways and the role of these signaling pathways in CB1R-mediated neurite outgrowth in Neuro-2A cells. The CB1 agonist HU-210 induced pertussis toxin-sensitive Src and Stat3 phosphorylation. Dominant negative (DN) mutants of Src and Stat3 blocked CB1R-induced neurite outgrowth. Constitutively active Rap 1B and Ral-activated Src and CB1R-induced Src phosphorylation was inhibited by Rap1-DN and Ral-DN, indicating that both Rap1 and Ral mediate downstream signaling from G alpha(o/i) for Src activation. Rap1-activated Ral and Ral-DN blocked Rap-induced Src phosphorylation. G alpha(o)-induced Stat3 activation was blocked by Ral-DN, whereas v-Src-induced Stat3 activation was not inhibited by Ral-DN, indicating that the CB1R, through G alpha(o), mediates the sequential activation of Rap1 to Ral to Src to Stat3 in Neuro-2A cells. Downstream of Src, the CB1R also activated Rac1 and JNK, which enhanced CBR1-mediated Stat3 activation. Rac-DN blocked CB1R-induced activation of JNK. Pharmacological inhibition of JNK blocked Src and CB1R activation of Stat3, indicating that Rac and JNK are also involved in CB1R-mediated neurite outgrowth. Overall, this study demonstrated that G alpha(o/i)-coupled CB1R triggers neurite outgrowth in Neuro-2A through the activation of a signaling network containing two pathways that bifurcate at Src and converge at Stat3.

Animals↗
PubMed2005

Neuropeptide processing profile in mice lacking prohormone convertase-1.

Prohormone convertase 1 (PC1; also known as PC3) is believed to be responsible for the processing of many neuropeptide precursors. To look at the role PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined changes in the levels of multiple neuropeptide products in PC1 knockout (KO) mice. The processing of proenkephalin was impaired in PC1 KO mouse brains with a decrease in the level of Met-Enkephalin immunoreactivity (ir-Met-Enk) and an accumulation of higher molecular weight processing intermediates containing ir-Met-Enk. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains with a decrease in the level of an endogenous 3 kDa C-terminal peptide. In contrast, the processing of proSAAS into PEN was not altered in PC1 KO mouse brains. Quantitative mass spectrometry was used to analyze a number of peptides derived from proopiomelanocortin (POMC), provasopressin, prooxytocin, chromogranin A, chromogranin B, and secretogranin II. Among them, the levels of oxytocin and peptides derived from chromogranin A and B dramatically decreased in the PC1 KO mouse pituitaries, while the levels of peptides derived from proopiomelanocortin and provasopressin did not show substantial changes. In conclusion, these results support the notion that PC1 plays a key role in the processing of multiple neuroendocrine peptide precursors and also reveal the presence of a redundant system in the processing of a number of physiologically important bioactive peptides.

Amino Acid Sequence↗
PubMed2005

Teaching resources. Cell signaling systems: a course for graduate students.

This course uses the primary literature to develop a systems-level understanding of the information flow through the various cell signaling pathways and networks. Current areas of research activities in the experimental and theoretical understanding of cell signaling research are highlighted. This Teaching Resource describes a sample syllabus and organization for this course. Each lecture is 2 hours. After each set of lectures, there is a 2-hour discussion period during which students present an in-depth analysis of a primary literature article, selected in consultation with the lecturer.

Curriculum↗
PubMed2005

Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G(alpha)o/i-triggered proteasomal degradation of Rap1GAPII.

The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth.

Amino Acid Sequence↗
PubMed2005

Mechanisms of action of antidepressants: from neurotransmitter systems to signaling pathways.

Antidepressants are commonly used in the treatment of anxiety and depression, medical conditions that affect approximately 17-20% of the population. The clinical effects of antidepressants take several weeks to manifest, suggesting that these drugs induce adaptive changes in brain structures affected by anxiety and depression. In order to develop shorter-acting and more effective drugs for the treatment of anxiety and depression, it is important to understand how antidepressants bring about their beneficial effects. Recent reports suggest that antidepressants can induce neurogenesis in the adult brain, although the mechanisms involved are not clearly understood. In this review, we describe the different neurotransmitter systems that are affected by anxiety and depression and how they are modulated by antidepressant treatment with a focus on signaling molecules and pathways that are activated during neurotransmitter receptor induced neurogenesis.

Animals↗
PubMed2005

Embryonic gene expression and pro-protein processing of proSAAS during rodent development.

In vitro assays have demonstrated that peptides derived from the recently-identified proSAAS precursor inhibit prohormone convertase 1 (PC1) suggesting that this novel peptide may function as an endogenous inhibitor of PC1. To further understand the role of proSAAS in vivo, we have investigated the expression of proSAAS mRNA and processing of proSAAS during pre- and early postnatal rodent development. In situ hybridization showed that, by embryonic day 12.5 (e12.5) in the rat, proSAAS mRNA was present in essentially all differentiating neurons in the mantle layer of the myelencephalon, metencephalon, diencephalon, spinal cord and several sympathetic ganglia. During later stages of prenatal development, widespread proSAAS expression continues in post-mitotic neurons of both the CNS and PNS and begins in endocrine cells of the anterior and intermediate pituitary. Although proSAAS expression overlaps with PC1 in several regions, its overall expression pattern is significantly more extensive, suggesting that proSAAS may be multifunctional during development. Processed forms of proSAAS are present by at least mid-gestation with marked accumulation of two C-terminal forms, comprising the PC1 inhibitory fragment of proSAAS.

Animals↗
PubMed2004

A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.

Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, delta opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis for these observations is not understood. We propose the modulation of receptor function by physical association between mu and delta opioid receptors as a potential mechanism. In support of this hypothesis, we show that mu-delta interacting complexes exist in live cells and native membranes and that the occupancy of delta receptors (by antagonists) is sufficient to enhance mu opioid receptor binding and signaling activity. Furthermore, delta receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus, heterodimeric associations between mu-delta opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies.

Analgesia↗
PubMed2004

Interactions between delta opioid receptors and alpha-adrenoceptors.

1. Several studies have reported functional interactions between different subtypes of opioid and alpha2A-adrenoceptors in the induction of spinal cord analgesia. The mechanisms underlying this phenomenon are not well characterized. We propose that direct receptor-receptor associations could account for some of the observed functional interactions. In the present study, we examined the presence of delta opioid receptors and alpha2A-adrenoceptors in interacting complexes and the functional implications of such interactions on receptor activity. 2. Using the proximity based bioluminescence resonance energy transfer (BRET) assay, we found that the delta opioid receptors and alpha2A-adrenoceptors are in close enough proximity (< 100 A) in live cells that can foster physical interactions. 3. Using coimmunoprecipitation of differentially epitope-tagged receptors, we found that delta opiate receptors exist in interacting complexes with alpha2A-adrenoceptors in heterologous cells. 4. Finally, using receptor activity mediated neurite outgrowth in Neuro 2A cells as a physiological readout, we found that interactions between delta opiate receptors and alpha2A-adrenoceptors have functional consequences. The expression of alpha2A-adrenoceptors is sufficient to promote delta opiate receptor-mediated neurite outgrowth, suggesting that the presence of inactive alpha2A-adrenoceptors can enhance delta opiate receptor-mediated signalling. 5. Taken together, these findings suggest that modulation of receptor function as a result of physical associations between delta opiate receptors and alpha2A-adrenoceptors may account for the observed synergy between opiate and adrenergic agonists in spinal analgesia.

Blotting, Western↗
PubMed2004

Obesity and diabetes in transgenic mice expressing proSAAS.

ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.

Animals↗
PubMed2004

Endothelin converting enzyme-2: a processing enzyme involved in the generation of novel neuropeptides.

Members of several metalloprotease families have been proposed to be involved in non-classical processing of neuroendocrine precursors. Among them, endothelin converting enzyme-2 (ECE-2) is a good candidate since it exhibits a neuroendocrine distribution, intracellular subcellular localization, and an acidic pH optimum. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. These results are consistent with an important role for ECE-2 in the processing of regulatory peptides at non-classical sites.

Amino Acid Sequence↗
PubMed2003

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides.

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.

Amino Acid Sequence↗
PubMed2002

Processing of proSAAS in neuroendocrine cell lines.

ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse-chase analysis using [(3)H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60-80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides.

Animals↗
PubMed2002

Oligomerization of opioid receptors.

Opioid receptors belong to the family of G-protein-coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by agonists such as morphine and endogenous opioid peptides leads to the activation of inhibitory G-proteins followed by a decrease in the levels of intracellular cAMP. Opioid receptor activation is also associated with the opening of K(+) channels and the inhibition of Ca(2+) channels. A number of investigations, prior to the development of opioid receptor cDNAs, suggested that opioid receptor types interacted with each other. Early pharmacological studies provided evidence for the probable interaction between opioid receptors. More recent studies using receptor selective antagonists, antisense oligonucleotides, or animals lacking opioid receptors further suggested that interactions between opioid receptor types could modulate their activity. We examined opioid receptor interactions using biochemical, biophysical, and pharmacological techniques. We used differential epitope tagging and selective immunoisolation of receptor complexes to demonstrate homotypic and heterotypic interactions between opioid receptor types. We also used the proximity-based bioluminescence resonance energy transfer assay to explore opioid receptor-receptor interactions in living cells. In this article we describe the biochemical and biophysical methods involved in the detection of receptor dimers. We also address some of the concerns and suggest precautions to be taken in studies examining receptor-receptor interactions.

Animals↗
PubMed2002

Developmental changes in opioid peptides and their receptors in Cpe(fat)/Cpe(fat) mice lacking peptide processing enzyme carboxypeptidase E.

Carboxypeptidase E (CPE) is involved in the biosynthesis of a number of neuropeptides including opioid peptides. A point mutation in this gene results in a loss of enzyme activity, decrease in mature neuroendocrine peptides, and development of late onset obesity as seen in Cpe(fat)/Cpe(fat) mice. In this study, we examined the processing of peptides derived from prodynorphin and proenkephalin in various brain regions of these mice during development. At 6 to 8 weeks, an age prior to the onset of obesity, levels of dynorphin peptides are decreased in all brain regions, whereas levels of ir-Met-enkephalin are differentially altered. There is an accumulation of C-terminally extended forms of all three opioid peptides in Cpe(fat)/Cpe(fat) mice, consistent with a lack of CPE activity. Thus, it appears that there is no direct correlation between the level of mature opioid peptides and the development of obesity in these mice. Since altered levels of peptides can influence the opioid receptor system, we examined the functional activity of mu and kappa opioid receptors using [35S]guanosine-5'-O-(gamma-thio)-triphosphate binding assays. We find no differences in kappa receptor activity in Cpe(fat)/Cpe(fat) compared with control littermate mice. In contrast, the mu receptor activity is differentially altered in select regions of Cpe(fat)/Cpe(fat) mice in response to a mu-specific ligand. Taken together, these results suggest that the lack of CPE activity leads to alterations in the level of opioid peptides during development and that changes in peptide levels differentially affect opioid receptor activity in vivo.

Animals↗