Decreased GyBAA Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino outyric acid is a major inhibitory neurotrarsr titter in the central nervous system. In the preset study sv, Have investigate(' the alteration of GABA receptor, In t he hrain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 It and 7 days partially pancreatectonnsea. GABA was (juan- (ified by [H]GABA receptor iispiacement method. GABA receptor kin: 10, pat at i et•ers were studied by using the binding of F'.](iAhA as ligand to the Triton X-100 treated me,i1,;-:mes a1,J displacement with unlabelled GABA. GhRA,v receptor activity was studied by using the [` -1 h3cuculline and displacement with unlabellecV euculline. ;.\13A content significantly decreased (1' < (1.(101 ) it, 0-e brain stern during the regeneration of pancreas. 'I hl, high affinity (IAI3A receptor binding sho?:ed it sigii'f cant decrease in 131„.,\ (P < 11.01) and K,I 1).05) n 72 h and 7 days after partial pancreatee 'timv. ";:flhicuculline hin(Iing showed it signih eat, 'le ( r(, :,e in /Jn1,s and K,I (P < 0.001) in 72 h pa^.rcreaw,, mised rats when compared with sham wt--tt' as P,n and K,I reversed to near sham after 7 da,s of pancreatectomv. The results sugge,) that GAB A throur,r; ('GABA receptors in brain Atcem has a regulatory uie during active regeneration of pancreas which will have inunense clinical significance in the treatment of cliahetcs.

Brain 5HT2A Receptor Regulation by Tryptophan Supplementation in Streptozotocin Diabetic Rats

5-HT2A receptor binding parameters were studied in the cerebral cortex and brain stem of control, diabetic, insulin, insulin + tryptophan and tr3yptophan treated streptozotocin diabetic rats. Scatchard analysis using selective antagonist, [-H](±)2,3-dimethoxyphenyl-l-[2-(4-piperidine)- methanol] ([3H]MDL100907) in cerebral cortex of diabetic rats showed a significant decrease in dissociation constant (Kd) without any change in maximal binding (Bm). Competition binding studies in cerebral cortex using ketanserin against [3H]MDL100907 showed the appearance of an additional site in the low affinity region during diabetes. In the brain stem, Scatchard analysis showed a significant increase in Bmax and Kd. Displacement studies showed a shift in the receptor affinity towards a low affinity state. All these altered parameters in diabetes were reversed to control level by insulin, insulin + tryptophan and tryptophan treatments. Tryptophan treatment is suggested to reverse the altered 5-HT2Abinding and blood glucose level to control status by increasing the brain 5-HT content.

GABAA and GABAB Receptor Gene Expression and Functional Regulation During Pancreatic Regeneration and Insulin Secretion in Rats

The present study demonstrate the functional alterations of the GABAA and GABAB receptors and the gene expression during the regeneration of pancreas following partial pancreatectomy. The role of these receptors in insulin secretion and pancreatic DNA synthesis using the specific agonists and antagonists also are studied in vitro. The alterations of GABAA and GABAR receptor function and gene expression in the brain stem, crebellum and hypothalamus play an important role in the sympathetic regulation of insulin secretion during pancreatic regeneration. Previous studies have given much information linking functional interaction between GABA and the peripheral nervous system. The involvement of specific receptor subtypes functional regulation during pancreatic regeneration has not given emphasis and research in this area seems to be scarce. We have observed a decreased GABA content, down regulation of GABAA receptors and an up regulation of GABAB receptors in the cerebral cortex, brain stem and hypothalamus. Real Time-PCR analysis confirmed the receptor data in the brain regions. These alterations in the GABAA and GABAB receptors of the brain are suggested to govern the regenerative response and growth regulation of the pancreas through sympathetic innervation. In addition, receptor binding studies and Real Time-PCR analysis revealed that during pancreatic regeneration GABAA receptors were down regulated and GABAB receptors were up regulated in pancreatic islets. This suggests an inhibitory role for GABAA receptors in islet cell proliferation i.e., the down regulation of this receptor facilitates proliferation. Insulin secretion study during 1 hour showed GABA has inhibited the insulin secretion in a dose dependent manner in normal and hyperglycaemic conditions. Bicuculline did not antagonize this effect. GABAA agonist, muscimol inhibited glucose stimulated insulin secretion from pancreatic islets except in the lowest concentration of 1O-9M in presence of 4mM glucose.Musclmol enhanced insulin secretion at 10-7 and 10-4M muscimol in presence of 20mM glucose- 4mM glucose represents normal and 20mM represent hyperglycaemic conditions. GABAB agonist, baclofen also inhibited glucose induced insulin secretion and enhanced at the concentration of 1O-5M at 4mM glucose and at 10-9M baclofen in presence of 20mM glucose. This shows a differential control of the GABAA and GABAB receptors over insulin release from the pancreatic islets. During 24 hours in vitro insulin secretion study it showed that low concentration of GABA has inhibited glucose stimulated insulin secretion from pancreatic islets. Muscimol, the GABAA agonist, inhibited the insulin secretion but, gave an enhanced secretion of insulin in presence of 4mM glucose at 10-7 , 10-5 and 1O-4M muscimol. But in presence of 20mM glucose muscimol significantly inhibited the insulin secretion. GABAB agonist, baclofen also inhibited glucose induced insulin secretion in presence of both 4mM and 20mM glucose. This shows the inhibitory role of GABA and its specific receptor subtypes over insulin synthesis from pancreatic bete-islets. In vitro DNA synthesis studies showed that activation of GABAA receptor by adding muscimol, a specific agonist, inhibited islet DNA synthesis. Also, the addition of baclofen, a specific agonist of GABAB receptor resulted in the stimulation of DNA synthesis.Thus the brain and pancreatic GABAA and GABAB receptor gene expression differentially regulates pancreatic insulin secretion and islet cell proliferation during pancreatic regeneration. This will have immense clinical significance in therapeutic applications in the management of Diabetes mellitus.

Dopamine Receptor Subtypes,IP3, cAMP and cGMP Functional Regulation in Parkinsonism Induced by Unilateral Infusion of Rotenone in Rats: Recovery with Bone Marrow Cells,Serotonin and GABA Supplementation

In the present study, the effects of 5-HT, GABA and Bone Marrow Cells infused intranigrally to substantia nigra individually and in combinations on unilateral rotenone infused Parkinsonism induced rats. Scatchard analysis of DA, DA D1 and D2 receptors in the corpus striatum, cerebral cortex, cerebellum, brain stem and hippocampus showed a significant increase in the Brain regions of rotenone infused rat compared to control. Real Time PCR amplification of DA D1, D2, Bax and ubiquitin carboxy-terminal hydrolase were up regulated in the brain regions of rotenone infused rats compared to control. Gene expression studies of -Synuclien, cGMP and Cyclic AMP response element-binding protein showed a significant down regulation in Rotenone infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies.Our study demonstrated that BMC administration alone cannot reverse the above said molecular changes occurring in PD rat. 5-HT and GABA acting through their specific receptors in combination with bone marrow cells play a crucial role in the functional recovery of PD rats. 5-HT, GABA and Bone marrow cells treated PD rats showed significant reversal to control in DA receptor binding and gene expression. 5-HT and GABA have co-mitogenic property. Proliferation and differentiation of cells re-establishing the connections in Parkinson's disease facilitates the functional recovery. Thus, it is evident that 5-HT and GABA along with BMC to rotenone infused rats renders protection against oxidative, related motor and cognitive deficits which makes them clinically significant for cellbased therapy. The BMC transformed to neurons when co-transplanted with 5-HT and GABA which was confirmed with PKH2GL and nestin. These newly formed neurons have functional significance in the therapeutic recovery of Parkinson’s disease.

Adrenergic and Glutamergic Receptor Gene Expression and Their Functional regulation in the Cerebral Cortex of Hypoxia Induced Neonatal Rats:Role of Oxygen,Epinephrine and Glucose Supplementation

In the present work, the role of oxygen, epinephrine and glucose supplementation in regulating neurotransmitter contents, adrenergic and glutamate receptor binding parameters in the cerebral cortex of experimental groups of neonatal rats were investigated. The study of neurotransmitters and their receptors in the cerebral cortex and the EEG pattern in the brain regions of neonatal rats were taken as index for brain damage due to hypoxia, oxygen and epinephrine. Real-Time PCR work was done to confirm the binding parameters. Second messenger, cyclic Adenosine Monophosphate (cAMP) was assayed to find the functional correlation of the receptors. Behavioural studies were carried out to confirm the biochemical and molecular studies. The efficient and timely supplementation of glucose plays a crucial role in correcting the molecular changes due to hypoxia, oxygen and epinephrine. The addictive neuronal damage effect due to oxygen and epinephrine treatment is another important observation. The corrective measures from the molecular study brought to practice will lead to maintain healthy intellectual capacity during the later developmental stages, which has immense clinical significance in neonatal care.

Withania somnitera and withanolide a mediated restoration of AMPA and NMDA receptor function in pilocarpine induced temporal lobe epilepsy

The onset of spontaneous seizures triggers a cascade of molecular and cellular events that eventually leads to neuronal injury and cognitive decline. The present study investigated the effect of Withania somnifera (WS) root extract and Withanolide A (WA) in restoring behavioural deficit by inhibiting oxidative stress induced alteration in glutamergic neurotransmission. The subdued performance in behavioural tests shows impaired motor coordination and memory. Histopathological investigations revealed significant neuronal loss in hippocampus of epileptic rats indicating glutamate mediated excitotoxicity. The treatment with WS and WA restored behavioural deficit and ameliorated neuronal loss. An altered redox homeostasis leading to oxidative stress is a hallmark of TLE. The antioxidant potential was afflicted in epileptic rats, evident from altered activity of SOD and CAT, down regulation of SOD and GPX expression and enhanced lipid peroxidation. The antioxidant property of WS and WA restored altered antioxidant capacity. Alteration in GDH activity and down regulation of GLAST expression resulted in enhanced glutamate content in the brain regions. The metabolism of glutamate was altered in the form of down regulated GAD expression. The alteration in synthesis, transport and metabolism resulted in further increase of the glutamate concentration at the synapse leading to glutamate mediated excitotoxicity. The decreased NMDA and AMPA receptor binding and down regulated NMDA R1, NMDA 2B and AMPA (GluR2) mRNA expression indicated altered glutamergic receptor function. The treatment with WS and WA reversed altered glutamergic receptor function, synthesis, transport and metabolism. The enhanced levels of second messenger IP3 responsible for Ca2+ mediated toxicity was reversed after treatment with WS and WA. Neurotoxics concentration of glutamate resulted in up regulation of pro apoptotic factors Bax and Caspase 8 and down regulation of anti apoptotic factor Akt resulting in neuronal death. The treatment with WS and WA resulted in activation of Akt and down regulation of Bax and caspase 8 leading to blocking of apoptotic pathway. The treatment with WS and WA resulted in reduced seizure frequency and amelioration of associated alterations suggesting the therapeutic role of Withania somnifera in temporal lobe epilepsy

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Pharmacological characterisation of the agonist radioligand binding site of 5-HT2A, 5-HT2B and 5-HT2C receptors

In the present study we compared the affinity of various drugs for the high affinity "agonist-preferring" binding site of human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors stably expressed in monoclonal mammalian cell lines. To ensure that the "agonist-preferring" conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([H-3]-ketanserin for 5-HT2A receptor and [H-3]-mesulergine for 5-HT2B and 5-HT2C receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([I-125]-DOI for 5-HT2A receptor binding and [H-3]-5-HT for 5-HT2B and 5-HT2C receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the "agonist-preferring" subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT2 receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT2A, 5-HT2B and 5-HT2C receptors respectively). There remains, however, a lack of highly selective agonists. (-)DOI is potent and moderately selective for 5-HT2A receptors, BW723C86 has poor selectivity for human 5-HT2B receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT2C receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT2 receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT2 receptor pharmacology.

Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

Mathematical modelling of competitive LDL/VLDL binding and uptake by hepatocytes

Elevated levels of low-density-lipoprotein cholesterol (LDL-C) in the plasma are a well-established risk factor for the development of coronary heart disease. Plasma LDL-C levels are in part determined by the rate at which LDL particles are removed from the bloodstream by hepatic uptake. The uptake of LDL by mammalian liver cells occurs mainly via receptor-mediated endocytosis, a process which entails the binding of these particles to specific receptors in specialised areas of the cell surface, the subsequent internalization of the receptor-lipoprotein complex, and ultimately the degradation and release of the ingested lipoproteins' constituent parts. We formulate a mathematical model to study the binding and internalization (endocytosis) of LDL and VLDL particles by hepatocytes in culture. The system of ordinary differential equations, which includes a cholesterol-dependent pit production term representing feedback regulation of surface receptors in response to intracellular cholesterol levels, is analysed using numerical simulations and steady-state analysis. Our numerical results show good agreement with in vitro experimental data describing LDL uptake by cultured hepatocytes following delivery of a single bolus of lipoprotein. Our model is adapted in order to reflect the in vivo situation, in which lipoproteins are continuously delivered to the hepatocyte. In this case, our model suggests that the competition between the LDL and VLDL particles for binding to the pits on the cell surface affects the intracellular cholesterol concentration. In particular, we predict that when there is continuous delivery of low levels of lipoproteins to the cell surface, more VLDL than LDL occupies the pit, since VLDL are better competitors for receptor binding. VLDL have a cholesterol content comparable to LDL particles; however, due to the larger size of VLDL, one pit-bound VLDL particle blocks binding of several LDLs, and there is a resultant drop in the intracellular cholesterol level. When there is continuous delivery of lipoprotein at high levels to the hepatocytes, VLDL particles still out-compete LDL particles for receptor binding, and consequently more VLDL than LDL particles occupy the pit. Although the maximum intracellular cholesterol level is similar for high and low levels of lipoprotein delivery, the maximum is reached more rapidly when the lipoprotein delivery rates are high. The implications of these results for the design of in vitro experiments is discussed.

Ligand dissociation from estrogen receptor is mediated by receptor dimerization: Evidence from molecular dynamics simulations

Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17 beta-estradiol (E-2) and the selective ER modulator raloxifene (RAL)] from the human ER alpha ligand-binding domain in monomeric and dimeric forms. E-2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II`). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I`). Remarkably, ER dimerization strongly suppresses Paths II and II` for E-2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.

Structural modeling of high-affinity thyroid receptor-ligand complexes

Understanding the molecular basis of the binding modes of natural and synthetic ligands to nuclear receptors is fundamental to our comprehension of the activation mechanism of this important class of hormone regulated transcription factors and to the development of new ligands. Thyroid hormone receptors (TR) are particularly important targets for pharmaceuticals development because TRs are associated with the regulation of metabolic rates, body weight, and circulating levels of cholesterol and triglycerides in humans. While several high-affinity ligands are known, structural information is only partially available. In this work we obtain structural models of several TR-ligand complexes with unknown structure by docking high affinity ligands to the receptors` ligand binding domain with subsequent relaxation by molecular dynamics simulations. The binding modes of these ligands are discussed providing novel insights into the development of TR ligands. The experimental binding free energies are reasonably well-reproduced from the proposed models using a simple linear interaction energy free-energy calculation scheme.

Recognition by the Thyroid Hormone Receptor of Canonical DNA Response Elements

To shed more light on the molecular requirements for recognition of thyroid response elements (TRES) by thyroid receptors (TRs), we compared the specific aspects of DNA TRE recognition by different TR constructs. Using fluorescence anisotropy, we performed a detailed and hierarchical study of TR-TRE binding. This wits done by comparing the binding affinities of three different TR constructs for four different TRE DNA elements, including palindromic sequences and direct repeats (F2, PAL, DR-1, and DR-4) as well as their interactions with nonspecific DNA sequences. The effect of MgCl(2) on suppressing of nonselective DNA binding to TR was also investigated. Furthermore, we determined the dissociation constants of the hTR beta DBD (DNA binding domain) and hTR beta DBD-LBD (DNA binding and ligand binding domains) for specific TRES. We found that a minimum DNA recognition peptide derived from DBD (H1TR) is sufficient for recognition and interaction with TREs, whereas scrambled DNA sequences were unrecognized. Additionally, we determined that the TR DBD binds to F2, PAL, and DR-4 with high affinity and similar K(d) values. The TR DBD-LBD recognizes all the tested TRES but binds preferentially to F2, with even higher affinity. Finally, our results demonstrate the important role played by LBDs in modulating TR-DNA binding.

Inhibition Mechanism of Rat alpha(3)beta(4) Nicotinic Acetylcholine Receptor by the Alzheimer Therapeutic Tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer`s disease, on currents elicited by activation of rat alpha(3)beta(4) nAChR heterologously expressed in KX alpha(3)beta(4)R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 +/- 0.2 mu M and 4.3 +/- 1.3 for the channel opening equilibrium constant, Phi(-1). Experiments were performed to investigate whether tacrine is able to activate the alpha(3)beta(4) nAChR. Tacrine did not activate whole-cell currents in KX alpha(3)beta(4)R2 cells but inhibited receptor activity at submicromolar concentration. Dose response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 mu M. The increase of Phi(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.

Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.