A glucagon-secretin-like peptide stimulates the intrinsic nervous plexus of guinea pig gallbladder

The role of vasoactive intestinal peptide (VIP), as a possible neurotransmitter of the intrinsic nerve plexus in the guinea pig gallbladder, was investigated by monitoring spontaneous contractile activity. VIP receptor antagonist (4 Cl-D-Phe6, Leu 17)-VIP did not produce any effect on muscular tone and spontaneous activity, whereas (N-Ac-Tyr1, D-Phe2)-GRF-(1-29)-NH2, (14-GRF analog), which is known to stimulate digestive enzyme secretion by interacting with the VIP-preferring receptors, greatly increased the amplitude and frequency of waves as well as the muscular tone. Since VIP receptor antagonist acts selectively as a competitive antagonist for the action of VIP, we conclude that the gallbladder inhibitory intrinsic plexus neurotransmitter is not VIP, but a member of the glucagon-secretin family of peptides.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Role of phospholipids in the binding activity of vasoactive intestinal peptide receptors

Summary Phospholipase digestion of rat intestinal epithelial cell membranes was performed in order to study the influence of membrane phospholipids on the binding activity of VIP receptors. Phospholipases A₂ and C strougly (ED₅₀4×10^–2 and 4×10^–1 g/ml, respectively) and rapidly reduced¹²⁵I-VIP binding to membranes whereas phospholipase D was ineffective. This suggests an important role of both hydrophobic and hydrophilic groups of phospholipids on VIP receptor binding activity.This work was supported by INSERM (CRL 827017) and the Fondation pour la Recherche Médicale Française.     

Untersuchungen über den Einfluss verschiedener Kationen auf die ATP-Spaltung durch die Zytoplasmafraktion vom Plexus chorioideus des Rindes

Summary The cytoplasma fraction of the bovine choroid plexus epithelial cells was found to contain a considerable ATPase activity. The influence of Na⁺, K⁺, Li⁺, Rb⁺, Cs⁺, Co⁺⁺, Mn⁺⁺, Zn⁺⁺ and Fe⁺⁺⁺ on the activity of the Mg⁺⁺-dependent enzyme has been studied. The monovalent cations do not influence the enzymic activity, whereas the effect of the bi- and trivalent cations is characterized by an inhibition of the ATPase.     

Renal vascular reactivity to ATP in hyper- and hypothyroid rats

The effects of adenosine triphosphate (ATP) on the renal vasculature of isolated kidneys from control, hyper- and hypothyroid rats were characterized. ATP responsiveness was evaluated in basal tone and in raised tone (phenylephrine 10^–6 M) preparations. These responses were compared with those obtained with barium chloride or sodium nitroprusside (SNP), used respectively as nonreceptor agonists for vasoconstriction or vasodilation. In preparations at basal tone, ATP produced dose-related vasoconstriction, which was increased in hyperthyroid kidneys, and was severely attenuated in kidneys from hypothyroid rats. In raised tone preparations from control rats ATP produced a dual response: vasoconstriction at low doses, which declined with increasing doses to give way to vasodilator responses; biphasic responses were found in some kidneys. Hyperthroid kidneys showed increased pressor responses and a vasodilator response similar to those seen in kidneys from control rats. However, in hypothyroid kidneys the vasodilator response was abolished. The responses to barium chloride and to SNP were significantly increased and decreased in hyper- and hypothyroid kidneys, respectively; vasoconstrictor responses to SNP were also found in hypothyroid kidneys. Hence the abnormal responses to ATP observed in both thyroid dysfunctions may be partially explained by unspecific alterations in the contractile machinery of the renal vasculature in these kidneys. However, ATP responsiveness (vasoconstriction at low tone and vasodilation at raised tone) was more severely affected in hypothyroid kidneys, suggesting that purinergic (P_2X and P_2Y) receptor activity may be decreased in these organs.     

Regulation ofL-valine absorption by opioids interacting withμ-receptors in rabbit ileum

In intact tissue, [d-Ala²,MePhe⁴, Gly-ol⁵] enkephalin (10^–5 M;-ligand), diminsihed short-circuit current (I_sc) and increased water, Na⁺ and Cl^– net fluxes in vitro under open circuit conditions; it also inhibitedL-valine absorption andL-valine-dependent variations of short-circuit current (I_{sc, val}). Naloxone (10^–6 M) antagonized these effects. In the absence of the muscularis and myenteric plexus this enkephalin or morphine (-ligand) reduced I_sc and I_{sc, val}. These enkephalin effects occurred at different times. Different concentrations of enkephalin were tested for their effects on I_{sc, val}. [d-Ala²,d-Leu⁵] enkephalin (mainly a -ligand) significantly decreased I_sc but not I_{sc, val}. The reduction ofL-valine absorption does not depend on the effects on basal ion transport. Interaction of opioids with-receptors located in the submucosal plexus and/or in the epithelial cell accounts for this reduction. This enkephalin effect seems to be at least partially under the control of the myenteric plexus.     

Melatonin and the electron transport chain

Melatonin protects the electron transport chain (ETC) in multiple ways. It reduces levels of ·NO by downregulating inducible and inhibiting neuronal nitric oxide synthases (iNOS, nNOS), thereby preventing excessive levels of peroxynitrite. Both ·NO and peroxynitrite-derived free radicals, such as ·NO₂, hydroxyl (·OH) and carbonate radicals (CO₃·^?) cause blockades or bottlenecks in the ETC, by ·NO binding to irons, protein nitrosation, nitration and oxidation, changes that lead to electron overflow or even backflow and, thus, increased formation of superoxide anions (O₂·^?). Melatonin improves the intramitochondrial antioxidative defense by enhancing reduced glutathione levels and inducing glutathione peroxidase and Mn-superoxide dismutase (Mn-SOD) in the matrix and Cu,Zn-SOD in the intermembrane space. An additional action concerns the inhibition of cardiolipin peroxidation. This oxidative change in the membrane does not only initiate apoptosis or mitophagy, as usually considered, but also seems to occur at low rate, e.g., in aging, and impairs the structural integrity of Complexes III and IV. Moreover, elevated levels of melatonin inhibit the opening of the mitochondrial permeability transition pore and shorten its duration. Additionally, high-affinity binding sites in mitochondria have been described. The assumption of direct binding to the amphipathic ramp of Complex I would require further substantiation. The mitochondrial presence of the melatonin receptor MT₁ offers the possibility that melatonin acts via an inhibitory G protein, soluble adenylyl cyclase, decreased cAMP and lowered protein kinase A activity, a signaling pathway shown to reduce Complex I activity in the case of a mitochondrial cannabinoid receptor.     

ADP receptor P2Y13 induce apoptosis in pancreatic β-cells

Pancreatic β-cell loss represents a key factor in the pathogenesis of diabetes. Since the influence of purinergic signaling in β-cell apoptosis has not been much investigated, we examined the role of the ADP receptor P2Y₁₃ using the pancreatic insulinoma-cell line MIN6c4 as a model system. Real time-PCR revealed high expression of the ADP receptors P2Y₁ and P2Y₁₃. Adding the ADP analogue, 2MeSADP, to MIN6c4 cells induced calcium influx/mobilization and inhibition of cAMP production by activation of P2Y₁ and P2Y₁₃, respectively. 2MeSADP reduced cell proliferation and increased Caspase-3 activity; both these effects could be fully reversed by the P2Y₁₃ receptor antagonist MRS2211. We further discovered that blocking the P2Y₁₃ receptor results in enhanced ERK1/2, Akt/PKB and CREB phosphorylation mechanisms involved in β-cell survival. These results indicate that P2Y₁₃ is a proapoptotic receptor in β-cells as the P2Y₁₃ receptor antagonist MRS2211 is able to protect the cells from ADP induced apoptosis.     

VIP and histamine H2 receptor activity in human fetal gastric glands

Summary Vasoactive intestinal peptide (VIP, EC₅₀=6.4×10^–10 M) and histamine (EC₅₀=3×10^–6 M) activated the cyclic AMP generating system in gastric glands isolated from two human fetuses at 23 weeks gestation. Histamine antagonism by the H₂ receptor blockers cimetidine (Ki=0.35×10^–6 M) and ranitidine (ki=0.51×10^–7 M) clearly characterized the histaminic activation as being of the H₂ type. It is suggested that these two vasoactive hormones may operate as neurocrine/paracrine regulators of the differentiation and/or function of the human gastric mucosa in utero.     

IP<Subscript>3</Subscript> receptor signaling and endothelial barrier function

The endothelium, a monolayer of endothelial cells lining vessel walls, maintains tissue-fluid homeostasis by restricting the passage of the plasma proteins and blood cells into the interstitium. The ion Ca²⁺, a ubiquitous secondary messenger, initiates signal transduction events in endothelial cells that is critical to control of vascular tone and endothelial permeability. The ion Ca²⁺ is stored inside the intracellular organelles and released into the cytosol in response to environmental cues. The inositol 1,4,5-trisphosphate (IP₃) messenger facilitates Ca²⁺ release through IP₃ receptors which are Ca²⁺-selective intracellular channels located within the membrane of the endoplasmic reticulum. Binding of IP₃ to the IP₃Rs initiates assembly of IP₃R clusters, a key event responsible for amplification of Ca²⁺ signals in endothelial cells. This review discusses emerging concepts related to architecture and dynamics of IP₃R clusters, and their specific role in propagation of Ca²⁺ signals in endothelial cells.     

Cholinesterases are down-expressed in human colorectal carcinoma

The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G₂^A) and monomers (G₁^A) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A₁₂, G₄^H and PRiMA-containing G₄^A AChE forms, besides G₄^H, G₄^A and G₁^H BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and overstimulating muscarinic receptors. Received 19 May 2006; received after revision 5 June 2006; accepted 5 July 2006     

The human α2-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region

The human α₂-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn³⁹⁸-Lys⁴⁵²), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys⁴²², Lys⁴²⁹, Lys⁴³⁶, Lys⁴⁵²) are completely and two (Lys⁴⁰⁶, Lys⁴¹⁵) are partially conserved. Multiple Lys to Ala mutants of A2PIC were expressed in Escherichia coli and used in intrinsic fluorescence titrations with kringle domains K1, K4, K4 + 5, and K1 + 2 + 3 of human plasminogen. We were able to identify the C-terminal Lys⁴⁵² as the main binding partner in recombinant A2PIC (rA2PIC) constructs with isolated kringles. We could show a cooperative, zipper-like enhancement of the interaction between C-terminal Lys⁴⁵² and internal Lys⁴³⁶ of rA2PIC and isolated K1 + 2 + 3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr⁴⁴⁵ in the unique C-terminal segment revealed no influence on the binding affinity to kringle domains.     

Differences in membrane ion transport between hepatocytes from the periportal and the pericentral areas of the liver lobule

We studied the Na⁺/K⁺ pump, Na⁺/K⁺ ATPase activity, and oxygen consumption (QO₂) in hepatocytes isolated from the periportal (PH) and pericentral (CH) regions of the liver lobule, to provide an insight into the functional properties of these cells. Na⁺/K⁺ pump activity was determined using⁸⁶Rb⁺ (a functional analog of K⁺) and ouabain, a specific inhibitor of this transport system. Our results indicate the the Na⁺/K⁺, pump and Na⁺/K⁺ ATPase activity are significantly lower in CH than in PH, although basal ouabain-sensitive (OS) QO₂ was negligible in both of these cell preparations. However, OSQO₂ was significantly lower in CH than in PH when the Na⁺/K⁺ pump was activated using the ionophore nystatin in a Na⁺-containing medium. These results indicate that the differences in membrane ion transport exist between hepatocytes from different locations of the liver lobule.     

GPR39: a Zn<Superscript>2+</Superscript>-activated G protein-coupled receptor that regulates pancreatic,gastrointestinal and neuronal functions

GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn²⁺ has been shown to be a potent stimulator of GPR39 activity via the Gα_q, Gα_12/13 and Gα_s pathways. The potency and specificity of Zn²⁺ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn²⁺ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63 Ag8 cells

Summary Using the suspension cell line P3X63 Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (M_r 40 kD) and Semliki Forest virus core protein (M_r 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0°C. Subsequent resealing of the pores was completed after a 5-min incubation at 37°C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37°C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K⁺, Mg²⁺, and Ca²⁺) and resealing (in the presence of K⁺, Mg²⁺, and Ca²⁺), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

TRPM7 is regulated by halides through its kinase domain

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg²⁺), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg²⁺ ([Mg²⁺]_i) and this is facilitated through the ATP-binding site of the channel’s kinase domain. The synergistic block of TRPM7 by chloride and Mg²⁺ is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg²⁺]_i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.     

Development of a new tachykinin antagonist

Summary The mouse urinary bladder possesses a tachykinin receptor which responds to kassinin and eledoisin, but not to some other tachykinins. The action of kassinin, but not that of eledoisin, was blocked in a surmountable manner by a new tachykinin antagonist, D-Pro⁴, Val⁸, D-Trp^7,9,10 (SP)^4–11.Acknowledgments. We thank Mrs Danielle Solomos for technical help. This study was financed by a grant from the Swiss National Science Foundation (3.800-80.2).     

Old drugs with new skills: fenoprofen as an allosteric enhancer at melanocortin receptor 3

The efficiency of drug research and development has paradoxically declined over the last decades despite major scientific and technological advances, promoting new cost-effective strategies such as drug repositioning by systematic screening for new actions of known drugs. Here, we performed a screening for positive allosteric modulators (PAMs) at melanocortin (MC) receptors. The non-steroidal anti-inflammatory drug fenoprofen, but not the similar compound ibuprofen, presented PAM activity at MC₃, MC₄, and MC₅ receptors. In a model of inflammatory arthritis, fenoprofen afforded potent inhibition while ibuprofen was nearly inactive. Fenoprofen presented anti-arthritic actions on cartilage integrity and synovitis, effects markedly attenuated in Mc3r?/? mice. Fenoprofen displayed pro-resolving properties promoting macrophage phagocytosis and efferocytosis, independently of cyclooxygenase inhibition. In conclusion, combining repositioning with advances in G-protein coupled receptor biology (allosterism) may lead to potential new therapeutics. In addition, MC₃ PAMs emerged as a viable approach to the development of innovative therapeutics for joint diseases.     

Long-term administration of a substance P antagonist, (D-Pro2, D-Trp7,9)-SP,abolishes the response to ocular trauma

Summary The long-term effect of ocular administration of a substance P (SP) antagonist, (D-Pro², D-Trp^7,9)-SP, was studied in the rabbit. After 2–3 months of topical administration of the antagonist twice daily, a mild trauma was applied in the form of infrared irradiation of the iris. The control eye responded with disruption of the blood-aqueous barrier, the eye treated with the antagonist did not. Two days after termination of treatment, the response to ocular injury was still reduced. Another 2 days later, ocular injury evoked a normal response, which shows that the protection was reversible. No adverse reaction to the SP antagonist was noted.     

Inhibition of glucose transport in human erythrocytes by 2,3-dioxoindole (Isatin)

10 mM isatin (2,3-dioxoindole) inhibited glucose influx into human erythrocytes by over 30%. The inhibition is of the competitive type, where the affinity constant (K_t) was increased from 5.71 (control) to 11.11 mM in the presence of isatin with no change in V_max (130 nmol/min/ml packed cells). The observed inhibition of sugar transport by isatin was not mediated through membrane–SH groups accessible to iodoacetate, iodoacetamide, DTNB, DNP or sodium arsenite. Isatin inhibited sugar transport in the presence of 2 mM harmaline, an alkaloid inhibitor of Na⁺, K⁺–ATP_ase activity. The inhibition was non additive which suggests that these two compounds interact with the same or a similar site on the erythrocyte membrane.