Stimulated release of arachidonate and prostaglandins is vectorial in MDCK epithelial cells.

The receptor mediated activation of phospholipase A2 by appropriate ligands results in the synthesis and release of eicosanoids, a class of potent bioregulatory molecules. Madin-Darby canine kidney cells (MDCK) are polarized epithelial cells, with structurally and functionally distinct plasma membrane domains separated by tight junctions. Using MDCK cells grown in dual sided chambers, we show in this report, that a) the receptor mediated release of prostaglandins and arachidonate into the extracellular medium is predominantly unidirectional, b) the direction of release is agonist specific, and c) the magnitude of the response due to a given agonist is cell-domain specific. These characteristics, if operative in vivo, would contribute towards the optimal function of trans-cellular metabolism of eicosanoids already demonstrated.     

Shear stress-induced Ca2+ mobilization in MDCK cells is ATP dependent,no matter the primary cilium

Primary cilium has emerged as mechanosensor to subtle flow variations in epithelial cells, but its role in shear stress detection remains controversial. To probe the function of this non-motile organelle in shear stress detection by cells, we compared calcium signalling responses induced by shear stress in ciliated and unciliated MDCK cells. Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured using Fura-PE3 video imaging fluorescence microscopy in response to shear stress due to laminar flow (385 μl s^?1). Our results show that both unciliated and ciliated MDCK cells are shear stress sensitive via ATP release and autocrine feedback through purinergic receptors. However, purinergic calcium signals differed in response intensity and receptor subtypes. In unciliated cells, shear stress-induced elevation in [Ca²⁺]_i was predominantly mediated through P2X receptors (P2XR). In contrast, calcium mobilization in ciliated MDCK cells resulted from P2YRs and store-operated Ca²⁺-permeable channels besides P2XRs. These findings lend support to the hypothesis that ATP release in response to shear stress is independent of the primary cilium and that transduction of mechanical strain into a specific biochemical responses stems on the mobilization of different sets of purinergic receptors.     

Search for additional influenza virus to cell interactions

Sialyl oligosaccharides have long been considered to be the sole receptors for influenza virus. However, according to [1] some viruses are able to grow in sialic-free MDCK cells. Here we attempted to reveal a possible second, non-sialic receptor, hypothesizing the involvement of additional carbohydrate lectin recognition in influenza virus reception process, first of all in situations when a lectin of the host cell could recognize the viral carbohydrate ligand. We tested the presence of galactose- and sialic acid-binding lectins, as well as mannoside- and sulfo-N-acetyllactosamine-recognizing properties of MDCK and Vero cells using polyacrylamide neoglycoconjugates and antibodies. MDCK cells bind galactoside probes stronger than Vero cells, whereas Vero cells bind preferentially sialoside, mannoside and various sulfo-oligosaccharide probes. The probing of viruses with the neoglycoconjugates revealed specific 6′-HSO ₃ LacNAc (but not other sulfated oligosaccharides) binding property of A and B human strains. Affinity of 6′-HSO ₃ LacNAc probe was comparable with affinity of 6′-SiaLac probe but the binding was not inhibited by the sialooligosaccharide.     

TLR-4 mediated group IVA phospholipase A2 activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent

Group IVA phospholipase A₂ (GIVA PLA₂) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA₂. We also studied the effect of PAP-1 and PKC on Ca^+ 2 induced and synergy enhanced GIVA PLA₂ activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo₂-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca^+ 2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA₂ synergistic activation by stimulation with Kdo₂-Lipid A and ATP.     

Mitocryptide-2, a neutrophil-activating cryptide, is a specific endogenous agonist for formyl-peptide receptor-like 1

Peptides simultaneously produced during maturation and degradation of peptidergic hormones and functional proteins have recently become a great interest because they display unpredictably different biological roles than the parent proteins. Namely, we discovered two novel functional cryptic peptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial cytochrome c oxidase and cytochrome b, that efficiently induced neutrophilic migration and activation at nanomolar concentrations. We named these functional “cryptic” peptides hidden in protein structures as “cryptides.” In this study, we investigated the receptor molecules and cellular signaling mechanisms for neutrophil-activating N-formylated cryptide MCT-2. In order to identify the receptor molecules, we established HEK-293 cells stably expressing either formyl-peptide receptor (FPR) or its homologue FPR-like 1 (FPRL1), because neutrophilic cells express these receptor molecules which recognize N-formylated peptides. We observed that MCT-2 directly bound to FPRL1 and promoted an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and neither interacted with nor activated FPR, demonstrating that MCT-2 is a specific agonist for FPRL1. Moreover, MCT-2 induced not only [Ca²⁺]_i increase and phosphorylation of extracellular signal-regulated protein kinases 1 and 2, but also β-hexosaminidase release in neutrophilic/granulocytic cells differentiated from HL-60 cells. Such signaling events were diminished by pretreatment with pertussis toxin, indicating that MCT-2-promoted neutrophilic function is a consequence of G_i- or G_o-type G protein-dependent intracellular signaling events via FPRL1 activation. These findings suggest that MCT-2, a cryptide derived from mitochondrial cytochrome b, is a specific endogenous agonist for FPRL1 which is proposed to play key roles in inflammatory responses but whose physiological agonists are equivocal.     

Specific delta-opioid antagonists exert an agonist-independent inhibitory effect,similar to the agonist,on the release of GnRHIn Vitro

Summary 1. Inin vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissiset al., 1993).2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37°C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH.3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release.4. Diallyl-G also inhibits the release of prostaglandin E₂ (PGE₂). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE₂ but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release.5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.     

Adenosine receptor agonists affect taurine release from mouse brain stem slices in ischemia

The release of the inhibitory amino acid taurine is markedly enhanced under ischemic conditions in both adult and developing brain stem, together with a pronounced increase in the release of the neuromodulator adenosine. We now studied the effects of adenosine receptor agonists and antagonists on [³H]taurine release in the brain stem in normoxia and ischemia, using a superfusion system. Under standard conditions, the adenosine A₁ receptor agonist N⁶-cyclohexyladenosine (CHA) potentiated basal taurine release in adult mice, which response was blocked by the antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CHA and the A_2a receptor agonist 2-p-(2-carboxyethyl)phenylamino-5′-N-ethylcarboxaminoadenosinehydrochloride (CGS 21680) had no effect on the release in developing mice. In ischemia, CHA depressed both basal and K⁺-stimulated taurine release in developing mice in a receptor-mediated manner, blocked by DPCPX. The A_2a receptor agonist CGS 21680 was also inhibitory. Taurine and adenosine may thus not cooperate in developing mice to prevent ischemic neuronal damage. On the other hand, CGS 21680 enhanced taurine release in the adult brain stem in ischemia, both basal and K⁺-stimulated release being affected. These effects were abolished by the antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), indicating a receptor-mediated process. In this case elevated levels of taurine could be beneficial, protecting against hyperexcitation and excitotoxicity.     

Role of a putative tyrosine-O-sulfate receptor in the targeting and/or intracellular transport of tyrosine-sulfated proteins

By employing the affinity gel fraction technique, we have detected a 175 kDa tyrosine-O-sulfate (TyrS)-binding protein in sodium choleate extracts of the microsomal membrane fractions of bovine liver and pancreas, as well as canine liver and pancreas. Western blot analysis revealed the presence of the bovine liver TyrS-binding protein in complexes with tyrosine-sulfated proteins both in vivo and in vitro, suggesting the putative role of the former being the receptor for the latter. Using filter-grown Madin-Darby canine kidney (MDCK) cells as a model, it was demonstrated that the tyrosine-sulfated proteins synthesized were predominantly secreted into the apical medium. The results further indicate the production and differential polarized secretion of different sulfated forms of the two major secretory proteins produced by MDCK cells, fibronectin (FN) and an 80 kDa glycoprotein (gp 80), with their tyrosine-sulfated forms being predominantly secreted from the apical surface. Treatment of filter-grown MDCK cells with glycosylation inhibitors, swainsonine and 1-deoxymannojirimycin, appeared to enhance the apical secretion of tyrosine-sulfated FN and gp 80. A similar 175 kDa membrane-bound 'TyrS receptor', cross-reactive toward antiserum against the canine liver TyrS receptor, was shown to be present in MDCK cells. Pulse-chase experiments revealed its presence in complexes with newly synthesized FN and gp 80. A hypothetical model for TyrS residues serving as an apical targeting signal during the biosynthetic transport of tyrosine-sulfated proteins, as mediated by the TyrS receptor, in MDCK cells is proposed.     

The A3 adenosine receptor as a new target for cancer therapy and chemoprotection

Adenosine, a purine nucleoside, acts as a regulatory molecule, by binding to specific G-protein-coupled A₁, A_2A, A_2B, and A₃ cell surface receptors. We have recently demonstrated that adenosine induces a differential effect on tumor and normal cells. While inhibiting in vitro tumor cell growth, it stimulates bone marrow cell proliferation. This dual activity was mediated through the A3 adenosine receptor. This study showed that a synthetic agonist to the A3 adenosine receptor, 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide (Cl-IB-MECA), at nanomolar concentrations, inhibited tumor cell growth through a cytostatic pathway, i.e., induced an increase number of cells in the G0/G1 phase of the cell cycle and decreased the telomeric signal. Interestingly, Cl-IB-MECA stimulates murine bone marrow cell proliferation through the induction of granulocyte-colony-stimulating factor. Oral administration of Cl-IB-MECA to melanoma-bearing mice suppressed the development of melanoma lung metastases (60.8 ± 6.5% inhibition). In combination with cyclophosphamide, a synergistic anti-tumor effect was achieved (78.5 ± 9.1% inhibition). Furthermore, Cl-IB-MECA prevented the cyclophosphamide-induced myelotoxic effects by increasing the number of white blood cells and the percentage of neutrophils, demonstrating its efficacy as a chemoprotective agent. We conclude that A3 adenosine receptor agonist, Cl-IB-MECA, exhibits systemic anticancer and chemoprotective effects.     

Subtypes of Madin-Darby canine kidney (MDCK) cells defined by immunocytochemistry: Further evidence for properties of renal collecting duct cells

The Madin-Darby canine kidney (MDCK) cell line has been proposed as a model for studying intercalated (IC) cells of the renal cortical collecting duct. The IC cells are characterized by peanut lectin (PNA) binding capacity, carbonic anhydrase (CA) activity and Cl^-–HCO ₃ ^- exchange mediated by a band 3-related protein. It has been suggested that these properties are also expressed in MDCK cells. So far however, the nature of the specific protein involved in Cl^-–HCO ₃ ^- exchange, the type of CA isozyme and the relationship between these two characteristics and PNA binding, have not been investigated in MDCK cells by immunocytochemical methods. Using two antibodies raised against human erythrocyte band 3 protein and two against human erythrocyte CA I and II isozymes, our study provides evidence that a protein related to band 3 is expressed in about 5% of cultured MDCK cells; these band 3-positive cells do not bind PNA and are not reactive for CAI or CAII. About 30% of the MDCK cells bind PNA, two-thirds of which are also CAII-positive. A majority (about 65%) of MDCK cells is not reactive for the three markers used; their density is increased after incubation with aldosterone. These data indicate (i) that the Cl^-–HCO ₃ ^- exchanger of the MDCK cells could be related to human erythrocyte band 3, (ii) that the CA activity of the MDCK cell line bears antigenic identity with the erythrocyte CA II isozyme and (iii) that the latter is always co-localized with PNA binding. These results provide immunocytochemical evidence for the heterogeneity of the MDCK cell line, which might reflect the cellular heterogeneity encountered in the renal cortical collecting duct. Our data also indicate that clonal selection will be required for future functional studies of the MDCK cells.     

The D3 dopamine receptor inhibits dopamine release in PC-12/hD3 cells by autoreceptor signaling via PP-2B, CK1, and Cdk-5

The function of the D₃ dopamine (DA) receptor remains ambiguous largely because of the lack of selective D₃ receptor ligands. To investigate the function and intracellular signaling of D₃ receptors, we established a PC‐12/hD3 clone, which expresses the human D₃ DA receptor in a DA producing cell line. In this model, we find that the D₃ receptor functions as an autoreceptor controlling neurotransmitter secretion. Pre‐treatment with 3,6a,11, 14‐tetrahydro‐9‐methoxy‐2 methyl‐(12H)‐isoquino[1,2‐b] pyrrolo[3,2‐f][1,3] benzoxanzine‐1‐carboxylic acid, a D₃ receptor preferring agonist, dose‐dependently suppressed K⁺‐evoked [³H]DA release in PC‐12/hD3 cells but not in the control cell line. This effect was prevented by D₃ receptor preferring antagonists GR103691 and SB277011‐A. Furthermore, activation of D₃ receptors significantly inhibits forskolin‐induced cAMP accumulation and leads to transient increases in phosphorylation of cyclin‐dependent kinase 5 (Cdk5), dopamine and cAMP‐regulated phosphoprotein of M_r 32 000 and Akt. Because we observed differences in Cdk5 phosphorylation as well as Akt phosphorylation after DA stimulation, we probed the ability of Cdk5 and phosphatidylinositol‐3 kinase (PI3K) to influence DA release. Cdk5 inhibitors, roscovitine, or olomoucine, but not the PI3K inhibitor wortmannin, blocked the D₃ receptor inhibition of DA release. In a complimentary experiment, over‐expression of Cdk5 potentiated D₃ receptor suppression of DA release. Pertussis toxin, 3‐[(2,4,6‐trimethoxyphenyl)methylidenyl]‐indolin‐2‐one and cyclosporine A also attenuated D₃ receptor‐mediated inhibition of DA release indicating that this phenomenon acts through Gi/oα and casein kinase 1, and phosphatase protein phosphatase 2B (calcineurin), respectively. In support of previous data that D₃ DA receptors reduce transmitter release from nerve terminals, the current results demonstrate that D₃ DA receptors function as autoreceptors to inhibit DA release and that a signaling pathway involving Cdk5 is essential to this regulation.     

The calcium requirement in GnRH-stimulated LH release is not mediated through a specific action on receptor binding

A radioligand receptor assay was used to determine the effect of Ca⁺² and other cations on the binding of D-Ser-(t-Bu)⁶-desGly¹⁰EA GnRH (Buserelin acetate, Hoechst) to the gonadotropin releasing hormone receptor in rat pituitary membrane. Specific binding was reduced in the presence of CaCl₂, BaCl₂, MnCl₂, Co(CH₃COO)₂, MgCl₂, NaCl, or LaCl₃, although these salts have markedly different effects on receptor mediated gonadotropin release from intact cells. Consistent with this finding, compounds which chelate cations increased specific binding of the ligand. The decreased binding is explained solely by an effect on receptor affinity, which was reduced from 4 × 10⁹ M^?1 in the presence of 10 mM CaCl₂. Total receptor number was unchanged. These data suggest that the requirement for Ca⁺² in GnRH-stimulated LH release is not mediated through a specific action of this ion at the level of receptor binding.     

On the role of histamine receptors in regulating pigmentary responses in Oreochromis mossambicus melanophores

Purpose: The present work was carried out to reveal the involvement of histamine receptors at the neuro-melanophore junction of teleost, Oreochromis mossambicus.

Methods: The isolated scale melanophores were assayed using the mean melanophore size index and their responses were recorded in presence of various concentrations of histamine along with H₁ and H₂ receptor specific agonists and antagonist and potentiator compound 48/80.

Results: Melanophores showed high sensitivity to histamine and its specific agonists. Histamine caused a dose-dependent pigment aggregation, whereas 2-(2-Pyridyl) ethylamine (PEA), a specific H₁R agonist also caused aggregation in a similar manner. Conversely, amthamine, a specific H₂R agonist resulted in pigment dispersion. The effects were antagonized by mepyramine; specific H₁R antagonist and ranitidine a specific H₂R antagonist.

Conclusion: It is concluded that O. mossambicus melanophores have both H₁ and H₂ receptors which mediate melanophore aggregation and dispersion respectively. Compound 48/80 augmented the melanin-aggregating and dispersing effects of PEA and amthamine. It is suggested that the effect of histamine is directly mediated through H1 and H2 receptors, whereas H1Rs may be predominantly involved in the aggregatory responses.     

Minireview: Differential Targeting and Retention of G Protein-Coupled Receptors in Polarized Epithelial Cells

Abstract

Localization of receptors in discrete cellular microdomains undoubtedly contributes to their interaction with particular effectors and receptor targets. For G protein-coupled receptors, virtually nothing is known about the mechanisms and structural features responsible for their targeting to and retention in varying surface domains. We have shown that the G_j/G_o-coupled α_2A-adrenergic receptor (α_2AAR) is directly targeted to the lateral subdomain of MDCK II cells. Mutational analysis has revealed that regions in or near the bilayer are likely critical for α_2AAR targeting, whereas endofacial domains contribute to α_2AAR retention on the lateral surface. Although the α_2BAR also is enriched on the lateral subdomain at steady-state, its polarization occurs after initial random delivery to both apical and basolateral surfaces followed by a selective accumulation on the lateral subdomain. The α_2CAR also is expressed on the lateral subdomain and achieves its localization via direct delivery to the basolateral surface; however, the α_2CAR also exists in an as yet not fully characterized intracellular compartment. Interestingly, another G_j/G_o-coupled receptor, the A₁ adenosine receptor, is enriched on the apical surface of MDCK II calls and achieves this localization by direct apical delivery. These findings indicate that receptor delivery to polarized surfaces is not determined by receptor coupling to a specific subpopulation of G proteins.     

Transport-active renal tubular epithelial cells (MDCK and LLC-PK1) in culutre. Prostaglandin biosynthesis and its regulation by peptide hormones and ionophore

Renal tubular epithelial cells isolated from dog and pig kidney (MDCK and LLC-PK₁, respectively) transport water and electrolytes in culture. MDCK cells resemble collecting tubule cells by additional, but not all, morphologic and biochemical criteria. It has previously been reported that PGE₂ appears to regulate transport activity by MDCK cells as well as their proliferation. We investigated prostaglandin biosynthesis by MDCK and LLC-PK₁ cells and assessed the effects of peptide hormones, bradykinin and vasopressin, on the cells' prostaglandin biosynthesis. Thin-layer chromatography of radioactive products released by MDCK cells labelled with octatritiated of [¹⁴C] arachidonic acid indicated the presence of materials comigrating with PGE₂, PGI₂ (detected as 60oxo0PGF₁α) and PGF₂α, in decreasing order of abundance. Maclofenamate inhibited the biosynthesis of all radioactive peaks comigrating with PGs, thus confirming their identities as product of fatty acid cyclo-oxygenase activity. The chemical identities of [³H] PGE₂ and [³H] 6-oxo-PGF₁α made by the cells were further confirmed by treatment with KOH. Radioimmunoassay of culture fluids incubated with MDCK cells verified that PGE₂ was the most abundant prostaglandin. Tranylcypromine, thought to be a specific inhibitor of prostacyclic synthetase, inhibited PGE₂ as well as PGI₂ biosynthesis indicating a lack of specificity of the inhibitor. The observation of PGE₂ and PGF₂α as respectively the most and least abundant prostaglandinds made by MDCK was in disagreement with results from another laboratory in which the reverse order of abundance was found. This suggests the presence of more than one cell line identified as MDCK but having different biochemical properties.Bradykinin stimulated acylhydrolase activity as well as PGE₂ and PGI₂ biosynthesis in MDCK cells while vasopressin had little or no effect. These results support the hypothesis that bradykinin's natriuretic effects may be mediated by prostaglandinds and that vasopressin is unlikely to acutely stimulate prostaglandin biosynthesis in collecting tubule cells $\text{in}$$\text{vivo}$. Endogenous PGE₂ may also regulate the proliferation of MDCK cells in culture.In contrast to MDCK cells, LLC-PK₁ cells lacked significant prostaglandin biosynthetic capability as documented by radiometric thin-layer chromatography and radioimmunoassay. This suggests that prostaglandins may have a modulatory rather than an obligatory role in regulating transport activity by tubular epithelial cells.     

Control of protein translation by IP3R-mediated Ca2+ release in Drosophila neuroendocrine cells

The inositol 1,4,5-trisphosphate receptor (IP₃R) is one of two Ca²⁺ channels that gates Ca²⁺ release from ER-stores. The ligand IP₃, generated upon specific G-protein coupled receptor activation, binds to IP₃R to release Ca²⁺ into the cytosol. IP₃R also mediates ER-store Ca²⁺ release into the mitochondria, under basal as well as stimulatory conditions; an activity that influences cellular bioenergetics and thus, cellular growth and proliferation. In Drosophila neuroendocrine cells expressing a hypomorphic mutant of IP₃R, we observed reduced protein translation levels. Here, we discuss the possible molecular mechanism for this observation. We hypothesize that the cellular energy sensor, AMPK connects IP₃R mediated Ca²⁺ release into the mitochondria, to protein translation, via the TOR pathway.     

AGAP1/AP‐3‐dependent endocytic recycling of M5 muscarinic receptors promotes dopamine release

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M₅ is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M₅ and the AP‐3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP‐3 to M₅. Interaction with AGAP1 and activity of AP‐3 were required for the endocytic recycling of M₅ in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP‐3 or abrogation of AGAP1–M₅ interaction in vivo decreased the magnitude of presynaptic M₅‐mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor‐recycling pathway that may underlie mechanisms of G‐protein‐coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.     

Cholinoceptor-mediated control of catecholamine release from chromaffin cells in the American eel,Anguilla rostrata

The cholinergic agonist-induced secretion of catecholamines from chromaffin cells in the American eel, Anguilla rostrata, was assessed using a salineperfused posterior cardinal vein preparation. Direct membrane depolarization with 60 mmol·l^-1 K⁺ caused a significant release of catecholamines (adrenaline + noradrenaline) into the perfusate which was unaffected by pre-treatment with the ganglion blocker, hexamethonium (final concentration = 10^-3 mol · l^-1). The nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, evoked catecholamine release in response to several doses exceeding 10^-7 mol; at 10^-5 mol the response was abolished by pre-treatment with the ganglion blocker, hexamethonium (final concentration = 10^-3 mol · l^-1). The muscarinic receptor agonist, pilocarpine, did not elicit catecholamine release in response to any of the doses administered (10^-8–10^-4 mol). A single injection of the mixed nicotinic/muscarinic cholinoceptor agonist, carbachol (10^-5 mol), caused the release of catecholamines which was abolished by pre-treatment with hexamethonium but which was unaffected by pre-treatment with the muscarinic receptor antagonist atropine (final concentration = 10^-5 mol · l^-1). The results of this study indicate that the process of cholinergic agonist-induced catecholamine secretion from the chromaffin cells in the American eel is mediated exclusively by activation of nicotinic receptors with no involvement of the muscarinic receptor.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium iodide - MS222 ethylaminobenzoate - HPLC high-performance liquid chromatography - PCV posterior cardinal vein - SEM standard error of the mean     

Non-parenchymal cells as mediators of physiological responses in liver

Parenchymal cells (hepatocytes) are the sites at which the principal metabolic functions of the liver are located. In the perfused liver, responses (e.g. vasoconstriction and glycogenolysis) to stimulating agents such as zymosan, platelet-activating factor and arachidonic acid, are inhibited by indomethacin and bromophenacyl bromide, inhibitors of cyclo-oxygenase and phospholipase A₂, respectively. Since cultured Kupffer and endothelial cells but not hepatocytes, produce eicosanoids, and since eicosanoids and especially prostaglandins induce similar patterns of responses when added directly to the perfused liver, an involvement of these nonparenchymal cells in mediating the above responses is considered likely. We propose that in most situations the responses induced by these stimulating agents are mediated through a combination of pathways that include interaction of the agents directly with hepatocytes or with vasoactive cells (endothelial and/or smooth muscle cells), or interaction of agents initially with non-parenchymal cells to produce and release eicosanoids, which then subsequently interact with hepatocytes or with vasoactive cells.     

Activation of adenosine A2 receptors enhances high K+-evoked taurine release from rat hippocampus: A microdialysis study

Summary The present study was designed to examine which type of adenosine receptors was involved in enhancement of high K⁺-evoked taurine release fromin vivo rat hippocampus using microdialysis. Perfusion with 0.5 or 5.0 mM adenosine enhanced high K⁺-evoked taurine release. Perfusion with 2M R(–)-N⁶-2-phenylisopropyladenosine (PIA), a selective adenosine A₁ receptor agonist, did not modulate taurine release. Perfusion with 1M 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A₁ receptor antagonist, increased taurine release. On the other hand, perfusion with 20M 2-[4-(2-carboxyethyl)phenethylamino]-5-N-ethyl-carboxamideadenosine (CGS21680), a selective adenosine A_2A receptor agonist, enhanced taurine release, while perfusion with 1 mM 3,7-dimethyl-propagylxanthine (DMPX), an adenosine A₂ receptor antagonist, did not affect taurine release. These results demonstrate that adenosine enhances high K⁺-evoked taurine release via activation of adenosine A_2A receptors from both neurons and glial cells ofin vivo rat hippocampus.