Dopamine D2 receptors potentiate arachidonate release via activation of cytosolic, arachidonate-specific phospholipase A2

Several G(i)-linked neurotransmitter receptors, including dopamine D2 receptors, act synergistically with Ca(2+)-mobilizing stimuli to potentiate release of arachidonic acid (AA) from membrane phospholipids. In brain, AA and its metabolites are thought to act as intracellular second messengers, suggesting that receptor-dependent potentiation of AA release may participate in neuronal transmembrane signaling. To study the molecular mechanisms underlying this modulatory response, we have now used Chinese hamster ovary cells transfected with rat D2-receptor cDNA, CHO(D2). Two antisense oligodeoxynucleotides corresponding to distinct cDNA sequences of cytosolic, AA-specific phospholipase A2 (cPLA2) were synthesized and added to cultures of CHO(D2) cells. Incubation with antisense oligodeoxynucleotides inhibited D2 receptor-dependent release of AA but had no effect on D2-receptor binding or D2 inhibition of cyclic AMP accumulation. In addition, pharmacological experiments showed that D2 receptor-dependent AA release was prevented by nonselective phospholipase inhibitors (such as mepacrine) but not by inhibitors of membrane-bound, non-AA-specific PLA2 (such as p-bromophenacyl bromide). cPLA2 is expressed in brain tissue. The results, showing that cPLA2 participates in receptor-dependent potentiation of AA release in CHO(D2) cells, suggest that this phospholipase may serve a similar signaling function in brain.     

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype

1. We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 microM), PGE2 (0.1 nM-1 microM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2alpha and the stable thromboxane mimetic, U46619 (each at 1 microM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 microM) had no significant effect on [3H]-ACh release. 3. The selective TP-receptor antagonist, ICI 192,605 (0.1 microM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2alpha. 4. A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective) > PGE2 > M&B 28,767 (EP3 selective) > 17-phenyl-omega-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 microM), did not affect EFS-induced [3H]-ACh release. 5. AH6809 (10 microM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-omega-trinor PGE2 on [3H]-ACh release. 6. These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.     

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography

Ethanol disrupts signal transduction mediated by a variety of G-protein coupled receptors. We examined the effects of ethanol on arachidonic acid release mediated by muscarinic acetylcholine receptors. Chinese hamster ovary (CHO) cells transfected with the different subtypes of human muscarinic receptors (M1 to M5) were incubated with [3H]arachidonic acid ([3H]AA) for 18 hr, washed, and exposed to the cholinergic agonist carbamylcholine for 15 min. Carbamylcholine induced [3H]AA release from CHO cells expressing M1, M3, or M5, but not M2 or M4, muscarinic receptors. Dose response curves revealed that carbamylcholine stimulated [3H]AA release by up to 12-fold with an ECo of approximately 0.4 microM; maximal responses were obtained with 10 microM carbamylcholine. Exposure of M1-, M3-, or M5-expressing cells to ethanol for 5 min before stimulating with carbamylcholine reduced [3H]AA release by 40 to 65%; 50% of the maximal inhibition was obtained with an ethanol concentration of 30 to 50 mM. Ethanol did not affect basal [3H]AA release measured in the absence of carbamylcholine. Dose response curves suggest that ethanol acts as a noncompetitive inhibitor of muscarinic receptor-induced [3H]AA release insofar as maximal [3H]AA release was depressed in the presence of ethanol with no apparent change in the EC50 for stimulation by carbamylcholine. Exposure of CHO cells to 38 mM ethanol for 48 hr increased [3H]AA release induced by carbamylcholine without affecting basal [3H]AA release or altering the EC50 for carbamylcholine. These results indicate that ethanol acutely inhibits muscarinic receptor signaling through the arachidonic acid pathway in a noncompetitive manner, but chronically enhances muscarinic signaling through the same pathway.     

Potentiation of retinoic acid-induced differentiation of HL-60 cells by prostaglandin EP2 receptor

Human promyeloid HL-60 cells are differentiated by all-trans retinoic acid (RA) to granulocytes, and prostaglandin (PG) E2 potentiates the RA-induced differentiation. Here we examined which subtype of PGE receptors was involved in this potentiating activity of PGE2. Northern blot analysis demonstrated that HL-60 cells expressed three subtypes of PGE receptor, EP2, EP3, and EP4. Among various EP agonists, and EP2-selective agonist, butaprost, preferentially potentiated the RA-induced differentiation of HL-60 cells. Butaprost not only decreased the half-maximal concentration of RA but also increased the maximal level of the differentiation. Butaprost concentration-dependently stimulated the cAMP formation, and 8-Br-cAMP strongly potentiated the RA-induced differentiation. These results demonstrate that the EP2 receptor enhances the RA-induced differentiation of HL-60 cells via stimulation of adenylate cyclase.     

Direct dopamine D2-receptor-mediated modulation of arachidonic acid release in transfected CHO cells without the concomitant administration of a Ca2+-mobilizing agent

In CHO cells transfected with the rat dopamine D2 receptor (long isoform), administration of dopamine per se elicited a concentration-dependent increase in arachidonic acid (AA) release. The maximal effect was 197% of controls (EC50=25 nM). The partial D2 receptor agonist, (-)-(3-hydroxyphenyl)-N-n-propylpiperidine [(-)-3-PPP], also induced AA release, but with somewhat lower efficacy (maximal effect: 165%; EC50=91 nM). The AA-releasing effect of dopamine was counteracted by pertussis toxin, by the inhibitor of intracellular Ca2+ release, 8-(N N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), by excluding calcium from the medium, by the phospholipase A2 (PLA2) inhibitor, quinacrine, and by long-term pretreatment with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, it was antagonized by the D2 antagonists, raclopride and (-)-sulpiride--but not by (+)-sulpiride--and absent in sham-transfected CHO cells devoid of D2 receptors. The results obtained contrast to the previous notion that dopamine and other D2 receptor agonists require the concomitant administration of calcium-mobilizing agents such as ATP, ionophore A-23187 (calcimycin), thrombin, and TRH, to influence AA release from various cell lines.     

Type II secretory phospholipase A2 associated with cell surfaces via C-terminal heparin-binding lysine residues augments stimulus-initiated delayed prostaglandin generation

Type II secretory phospholipase A2 (sPLA2) has been shown to be induced by a variety of proinflammatory stimuli and, therefore, has been implicated in the inflammatory process. In order to determine whether association of sPLA2 with cell surfaces via heparan sulfate proteoglycan is important for its effects on cellular functions, we have identified the critical domain in sPLA2 for heparin and cell surface binding and examined its role in cellular prostaglandin (PG) biosynthesis. Replacement of several conserved Lys residues in the C-terminal region of mouse and rat sPLA2s by Glu resulted in a marked reduction of their capacities to bind to heparin and mammalian cell surfaces without affecting their enzymatic activities toward dispersed phospholipid as a substrate. CHO cells stably transfected with wild-type sPLA2 released about twice as much arachidonic acid (AA) during culture for 10 h with fetal calf serum and interleukin-1beta than cells transfected with vector alone, whereas the ability to enhance AA release was impaired in sPLA2 mutants incapable of binding to cell surfaces. AA released by wild-type sPLA2-transfected CHO cells was metabolized to prostaglandin E2 via prostaglandin endoperoxide H synthase (PGHS)-2 after IL-1beta stimulation, revealing a particular functional linkage of sPLA2 to PGHS-2. In contrast, A23187-initiated immediate AA release over 30 min was not affected by sPLA2 overexpression. Taken together, these results suggest that sPLA2 expressed endogenously and anchored on cell surfaces via its C-terminal heparin-binding domain is involved in the PGHS-2-dependent delayed PG biosynthesis initiated by growth factors and cytokines during long term culture.     

Study on the activation of phospholipases A2 by purinergic agonists in rat submandibular ductal cells

Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [3H]arachidonic acid ([3H]AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold. Both agonists also increased the release of [3H]AA from acini but at a lower level (+50% and +100% respectively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of [3H]AA but did not affect the release of [3H]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP. Only barium could partly substitute for calcium to restore the purinergic response. Zinc inhibited the release of [3H]AA. Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2). The iPLA2, not the calcium-dependent PLA2 (cPLA2), released [3H]oleic acid ([3H]OA) from RSMG ductal cells. It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA2 and cPLA2. Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.     

Analysis of inhibitory effects of scutellariae radix and baicalein on prostaglandin E2 production in rat C6 glioma cells

Inhibitory mechanism of the water extract of Scutellariae Radix on prostaglandin E2 (PGE2) release was examined in C6 rat glioma cells. Scutellariae Radix reduced a Ca2+ ionophore A23187-induced PGE2 release by inhibition of arachidonic acid (AA) liberation. Sho-saiko-to and San'o-shashin-to, which contain Scutellariae Radix, also inhibited PGE2 release. A23187 caused phosphorylation of mitrogen-activated protein kinase (MAPK), resulting in activation of cytosolic phospholipase A2 (cPLA2). Scutellariae Radix and baicalein inhibited the phosphorylation of MAPK. Baicalein, but not baicalin, inhibited A23187-induced PGE2 release. These results suggest that baicalein in Scutellariae Radix reduces AA liberation through the inhibition of the MAPK-cPLA2 pathway.     

Myocardial production of prostaglandins: its role in atrial natriuretic peptide release

In recent years, considerable evidence has been accumulated on prostaglandins (PG) in modulating atrial natriuretic peptide (ANP) release. In the current study we investigated whether eicosanoids promote isoproterenol-induced ANP secretion from superfused rabbit sliced atria. Inclusion of the cyclooxygenase inhibitor indomethacin (10 mumol) to the superfusing medium abolished isoproterenol-induced ANP release. Next, PGE2, but not PGF2 alpha or PGI2 (10 mumol), increased ANP release. Furthermore, isoproterenol-induced PGE2 formation was fully attenuated by indomethacin. Dibutyl-cAMP (0.5 mmol) had no effect on PGE2 formation, and the protein kinase A (PKA) inhibitor H89 (20 mumol) did not alter isoproterenol-induced PGE2 formation. On the other hand, indomethacin led to a significant decrease in isoproterenol-induced cAMP production. In addition, PGE2 enhanced basal cAMP concentration in superfusates. Superfusion of sliced atria by forskolin (10 mumol) or by dibutyl-cAMP (0.5 mmol) produced a significant rise in ANP release. Finally, H89 was ineffective on basal ANP release but abolished the increase of ANP release in response to isoproterenol or to PGE2. We conclude that: the effect of isoproterenol on ANP release is sensitive to indomethacin and H89; PGE2, but not PGE2 alpha or PGI2, increases ANP release; isoproterenol promotes myocardial PGE2 formation independently of adenylate cyclase and PKA activation pathways; and PGE2-induced ANP release is mediated by cAMP production and subsequent PKA activation. These results suggest that isoproterenol-induced ANP release appears to be mediated at least partly by PGE2 with underlying cAMP formation and PKA activation.     

Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells

This study investigated the signal transduction mechanisms of angiotensin-(1-7) [Ang-(1-7)]- and Ang II-stimulated arachidonic acid (AA) release for prostaglandin (PG) production in rabbit aortic vascular smooth muscle cells. Ang II and Ang-(1-7) enhanced AA release in cells prelabeled with [3H]AA. However, 6-keto-PGF1 alpha synthesis produced by Ang II was much less than that caused by Ang-(1-7). In the presence of the lipoxygenase inhibitor baicalein, Ang II enhanced production of 6-keto-PGF1 alpha to a greater degree than Ang-(1-7). Angiotensin type (AT)1 receptor antagonist DUP-753 inhibited only Ang II-induced [3H]AA release, whereas the AT2 receptor antagonist PD-123319 inhibited both Ang II- and Ang-(1-7)-induced [3H]AA release. Ang-(1-7), receptor antagonist D-Ala7-Ang-(1-7) inhibited the effect of Ang-(1-7), but not of Ang II. In cells transiently transfected with cytosolic phospholipase A2 (cPLA2), mitogen-activated protein (MAP) kinase or Ca(++)-/cal-modulin-dependent protein (CAM) kinase II antisense oligonucleotides, Ang-(1-7)- and Ang II-induced [3H]AA release was attenuated. The CaM kinase II inhibitor KN-93 and the MAP kinase kinase inhibitor PD-98059 attenuated both Ang-(1-7)- and Ang II-induced cPLA2 activity and [3H]AA release. Ang-(1-7) and Ang II also increased CaM kinase II and MAP kinase activities. Although KN-93 attenuated MAP kinase activity, PD-98059 did not affect CaM kinase II activity. Both Ang II and Ang-(1-7) caused translocation of cytosolic PLA2 to the nuclear envelope. These data show that Ang-(1-7) and Ang II stimulate AA release and prostacyclin synthesis via activation of distinct types of AT receptors. Both peptides appear to stimulate CaM kinase II, which in turn, via MAP kinase activation, enhances cPLA2 activity and release of AA for PG synthesis.     

Tumor necrosis factor-alpha inversely regulates prostaglandin D2 and prostaglandin E2 production in murine macrophages. Synergistic action of cyclic AMP on cyclooxygenase-2 expression and prostaglandin E2 synthesis

Increased synthesis of insulin-like growth factor-1 is induced in murine macrophages by prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNFalpha). Accordingly, we have investigated mechanisms regulating synthesis of PGE2 that might contribute to autocrine/paracrine effects on insulin-like growth factor-1 production. In response to zymosan, TNFalpha specifically induced a 5-fold increase in PGE2 synthesis, at the same time decreasing PGD2 production in a reciprocal fashion. Activators of cyclic AMP-dependent protein kinase (PKA), such as PGE2 itself or dibutyryl cyclic AMP, did not modify PGE2 production by themselves but potentiated the TNFalpha-induced increase in PGE2; this effect required both RNA and protein synthesis. No significant change in arachidonate release or production of other eicosanoids was observed. The inducible form of cyclooxygenase-2 (COX2) but not of the constitutive form COX1 was implicated in the generation of both PGE2 and PGD2 in these cells by use of specific inhibitors and effects of dexamethasone. Neither COX1 nor COX2 protein levels were affected by TNFalpha or PKA activators used alone, whereas in association, marked up-regulation of COX2 mRNA and protein was observed. Incubations of cells carried out with PGH2 demonstrated that PGE2 synthase activity was increased after a TNFalpha pretreatment. Taken together, our results suggest that TNFalpha induced a switch from the PGD2 to PGE2 synthesis pathway by regulating PGE2 synthase expression and/or activity and that activators of PKA markedly potentiated the TNFalpha-induced increase in PGE2 through up-regulation of COX2 gene expression.     

Cholera toxin B subunit activates arachidonic acid metabolism

Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E2), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin's activation of phospholipase A2, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT's known enzymatic activity (i.e., ADP-ribosylation of GSalpha), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc- murine Theta (Th)1.2-positive lymphoma cells deficient in GSalpha. The experimental results showed that native CT stimulated the release of [3H[AA from S49 cyc- cells at a level similar to that for S49 WT cells, indicating that GSalpha is not essential for this process. Further, levels of cAMP in the CT-treated cyc- cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [3H]AA metabolites from both S49 WT and cyc- cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [3H]AA from S49 cyc- cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.     

The emerging role of paclitaxel in breast cancer therapy

Bradykinin and thrombin caused a time- and dose-dependent stimulation of prostanoid biosynthesis in human dental-pulp fibroblasts, as assessed by the release of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of prostacyclin). The stimulatory effect of bradykinin and thrombin on PGE2 biosynthesis was maximal within 5-10 min. The concentration of bradykinin producing half-maximal stimulation (EC50) of PGE2 and prostacyclin formation was 10 nM. EC50 for thrombin-induced formation of PGE2 and prostacyclin were 0.05 and 0.2 U/ml, respectively. Bradykinin analogues with affinity to the bradykinin B2 receptor, but not those with affinity to the B1 receptor, caused a burst of PGE2 formation. The stimulatory action of bradykinin and thrombin on PGE2 biosynthesis was abolished by two structurally different cyclo-oxygenase inhibitors and significantly reduced by two corticosteroids. Thrombin dose-dependently enhanced the incorporation of [3H]-thymidine into DNA in pulpal fibroblasts by a mechanism that was unrelated to the effect on prostanoid biosynthesis. Bradykinin did not affect thymidine incorporation. Thrombin, but not bradykinin, stimulated the biosynthesis of type 1 collagen in the pulpal fibroblasts. The stimulatory effect of thrombin on collagen biosynthesis was not affected by cyclo-oxygenase inhibitors. These data show that human dental-pulp fibroblasts are equipped with receptors for bradykinin and thrombin linked to enhanced prostanoid biosynthesis. Occupancy of the thrombin receptors also leads to a prostaglandin-independent stimulation of cell proliferation and collagen biosynthesis.     

The foot in hereditary motor and sensory neuropathies in children

We investigated the regulation of COX-2 expression and activity by adenosine receptors in rat microglial cells. The selective adenosine A2a-receptor agonist CGS21680 and the non-selective adenosine A1- and A2-receptor agonist 5'-N-ethylcarboxiamidoadenosine (NECA) induced an increase in COX-2 mRNA levels and the synthesis of prostaglandin E2 (PGE2). The adenosine A1-receptor agonist cyclopentyladenosine (CPA) was less potent, and the adenosine A1-receptor-specific agonist N6-2-(-aminophenylo)ethyladenosine (APNEA) showed only marginal effects. Microglia expressed adenosine A1-, A2a-, and A3-, but not A2b-receptor mRNAs, whereas astroglial cells expressed adenosine A2b- but not A2a-receptor mRNA. The adenosine A2a-receptor selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) inhibited both CGS21680-induced COX-2 expression and PGE2 release. CGS21680-increased PGE2 levels were inhibited by dexamethasone, by the nonsteroidal antiinflammatory drug meloxicam, and by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CGS21680 and NECA both increased intracellular cAMP levels in microglial cells. Dibutyryl cAMP as well as forskolin induced the release of PGE2. The results strongly suggest that adenosine A2a-receptor-induced intracellular signaling events cause an up-regulation of the COX-2 gene and the release of PGE2. Apparently, the cAMP second messenger system plays a crucial role in COX-2 gene regulation in rat microglial cells. The results are discussed with respect to neurodegenerative disorders of the CNS such as Alzheimer's disease, in which activated microglia are critically involved and COX inhibitors may be of therapeutic benefit.     

No evidence for presynaptic opioid receptors on cholinergic, but presence of kappa-receptors on dopaminergic neurons in the rabbit caudate nucleus: involvement of endogenous opioids

The effects of various opioid receptor agonists and antagonists were studied in rabbit caudate nucleus slices preincubated with either [3H]dopamine or [3H]choline, superfused with medium (containing in most experiments the D2 receptor antagonist domperidone) and subjected to electrical field stimulation. The stimulation-evoked [3H]overflow from slices prelabeled with [3H]dopamine (evoked [3H]dopamine release) was significantly reduced by preferential kappa-opioid receptor agonists, like U-50,488 H, but not by mu- or delta-opioid receptor selective drugs. Opioid receptor antagonists shifted the concentration/response curve of U-50,488 H to the right (apparent pA2-value of the kappa-selective antagonist nor-binaltorphimine: 10.1) and enhanced the evoked dopamine release in the presence of a mixture of peptidase inhibitors. On the other hand, the [3H]overflow from rabbit caudate nucleus slices prelabeled with [3H]choline (evoked acetylcholine release) remained almost unaffected by any opioid receptor agonist, as long as the presynaptic D2 heteroreceptor was blocked with domperidone: in the absence of domperidone, U-50,488 H exhibited facilitatory effects. For comparison, the effects of the preferential delta-opioid receptor agonist DPDPE was also studied in slices of the rat striatum, where it clearly inhibited the evoked acetylcholine release. From our data we conclude that in the rabbit caudate nucleus the evoked dopamine release is inhibited by both exogenous and endogenous opioids via presynaptic kappa-opioid receptors, whereas the evoked release of acetylcholine is not, or only indirectly (via released dopamine) affected by opioids.     

A randomized study of combined spinal-epidural analgesia versus intravenous meperidine during labor: impact on cesarean delivery rate

We recently reported on the successful generation of immortalized (CEPI-17-CL4) cells from primary human corneal epithelial (P-CEPI) cells which exhibited phenotypic, immunohistochemical and metabolic characteristics akin to the P-CEPI cells. The aims of the present studies were to investigate the ligand binding and functional coupling of the histamine receptors to various biochemical and physiological systems in the P-CEPI and CEPI-17-CL4 cells and to relate these findings to the normal and/or pathophysiological role of histamine on the human ocular surface. Specific [3H]-pyrilamine binding to CEPI-17-CL4 cell homogenates comprised >93% of the total binding and represented interaction with an apparent single population of high affinity (Kd=3.76+/-0.78 nM; n=4) and saturable (Bmax = 1582+/-161 fmol g(-1) tissue) number of histamine-1 (H1) receptor binding sites on CEPI-17-CL4 cell homogenates. The H1-receptor selective antagonists, pyrilamine (Ki=3.6+/-0.84 nM, n=4) and triprolidine (Ki = 7.7+/-2.6 nM, n=3), potently displaced [3H]-pyrilamine binding, while the H2- and H3-receptor selective antagonists, ranitidine and clobenpropit, were weak inhibitors (K(i)s>13 microM). Histamine induced phosphoinositide (PI) hydrolysis 2.7-4.4 fold above basal levels and with a potency of 14.9+/-4.9 microM (n=9) and 4.7+/-0.2 microM (n=9) in P-CEPI and CEPI-17-CL4 cells, respectively. Histamine-induced PI turnover was antagonized by H1-receptor selective antagonist, triprolidine, with a potency (Ki) of 3.2+/-0.66 nM (n=10) and 3.03+/-0.8 nM (n=4) in P-CEPI and CEPI-17-CL4 cells, respectively, but weakly effected by 10 microM cimetidine and clobenpropit, H2- and H3-receptor antagonists. The PI turnover response was attenuated by pre-treatment of the cells with the selective phospholipase C inhibitor, U73122 (1-(6-((17beta-3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC50=4.8+/-2.4 microM, n = 3). Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI-17-CL4 cells with a potency of 6.3+/-1.5 microM (n=4). The histamine-induced [Ca2+]i mobilization was reduced by about 28% following pre-incubation of the cells with 4 mM EGTA. While triprolidine completely inhibited histamine-induced [Ca2+]i mobilization, it did not influence the bradykinin-induced [Ca2+]i mobilization response. Histamine (EC50s = 1.28-2.77 microM, n=3-4) concentration-dependently stimulated the release of interleukin-6 (IL-6), IL-8 and granulocyte macrophage colony-stimulating factor, but it did not significantly alter release of tumour necrosis factor-alpha, PGE2 or collagenase-1 (matrix metalloproteinase-1; MMP-1) from CEPI cells. However, IL-1 (10 ng ml(-1)), foetal bovine serum (10%) and phorbol-12-myristate-13-acetate (3 microg ml(-1)) were effective positive control secretagogues of all the cytokines, PGE2 and MMP-1, respectively, from these cells. It is concluded that the CEPI cells express H1-histamine receptors which are positively coupled to PI turnover and [Ca2+]i mobilization which may be directly or indirectly responsible for the release of various cytokines from these cells at physiologically and/or pathologically relevant concentrations.     

Desmoplastic cerebral astrocytoma of infancy: a case report

Strips of human right atrial appendages were preincubated with [3H]noradrenaline and then superfused with physiological salt solution containing inhibitors of uptake1 and uptake2. Tritium overflow was evoked by transmural electrical stimulation (standard frequency: 2 Hz). Prostaglandin E2 (PGE2) inhibited the electrically evoked tritium overflow; at the highest concentration investigated, tritium overflow was reduced by about 80% and the pIC50% value was 7.14. The effect of PGE2 was not affected by rauwolscine, which, by itself, increased the evoked overflow. Naproxen failed to affect the evoked tritium overflow and its inhibition by PGE2. The inhibitory effect of PGE2 on the electrically evoked tritium overflow was mimicked by prostaglandin E1, the EP1/EP3-receptor agonist sulprostone and the EP2/EP3-receptor agonist misoprostol with the rank order of potency (pEC50%): sulprostone (7.68) > misoprostol (7.10) > PGE1 (6.39). In contrast, PGF2alpha, the IP/EP1-receptor agonist iloprost and the stable thromboxane A2 analogue U46619 (9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin++ + F2alpha) did not change evoked tritium overflow. PGD2 caused facilitation. These results suggest that the sympathetic nerve fibres innervating human atrial appendages are endowed with presynaptic inhibitory EP3 and facilitatory DP-receptors. The EP3-receptors appear not to be tonically activated and do not interact with the alpha2-autoreceptors.     

D4 dopamine receptor-mediated signaling events determined in transfected Chinese hamster ovary cells

A Chinese hamster ovary (CHO) cell line stably expressing a recombinant human D4 dopamine receptor made from a synthetic gene has been used to determine potential D4-mediated signaling events. We designed and synthesized a modified gene coding for a human D4 receptor with reduced G + C content but unaltered encoded amino acids. Stable expression of this gene was obtained in two cell lines, inducible expression in CHO lacI cells and constitutive expression in HEK293 cells. In CHO lacI cells induced to express D4 receptors but not in uninduced cells, dopamine and quinpirole inhibit forskolin-stimulated cAMP accumulation and potentiate ATP-stimulated [3H]arachidonic acid release through a mechanism that requires protein kinase C but is unaffected by membrane-soluble cAMP analogs. In addition, D4 receptor activation causes an increase in the rate of extracellular acidification measured by microphysiometry. This response is unaffected by protein kinase C down-regulation but is inhibited by removal of extracellular sodium and inhibitors of NaH-1 exchange, suggesting the involvement of a Na+/H+ exchanger. All responses are blocked by clozapine and are sensitive to pertussis toxin. D4 receptors, like other G(i)/G(o)-linked receptors, mediate multiple signaling events, and the pathways activated are similar to those used by D2 and D3 receptors expressed in similar cells.     

Lack of direct connection between arachidonic acid release and prostanoid synthesis upon differentiation of U937 cell

The changes in AA incorporation and release as well as prostanoid synthesis upon differentiation of human premonocytic cell line, U937, induced by three functionally diverse agents--phorbol ester (TPA), dimethyl sulfoxide (DMSO), and retinoic acid (RA) have been investigated. The rate of AA incorporation into the cells remained unchanged whereas a 3- to 6-fold increase in AA release upon stimulation with Ca(2+)-ionophore A23187 as compared to undifferentiated cells was observed. While undifferentiated cells were incapable to metabolise AA via the cyclooxygenase pathway all three types of differentiated U937 cells produced TxB2 and PGE2. Only TPA-differentiated cells responded with a 6-fold increase of prostanoid synthesis after A23187 stimulation, whereas in DMSO-differentiated cells prostanoid synthesis was slightly stimulated by A23187 and in RA-differentiated cells it was not stimulated at all. Thus, agonist-induced prostanoid synthesis in differentiated cells is dependent on the nature of differentiating agent and does not correlate with AA liberation.