Endothelin- and Sarafotoxin-Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells

Abstract: The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphates (IPs) and tracheal smooth muscle contraction. BQ-123, an ET_A receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pK_B values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca²⁺ and was blocked by treatment with EGTA. The addition of Ca²⁺(3–620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca²⁺-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 μM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to down regulation of PKC. Inactive phorbol ester, 4α-phorbol 12, 13-didecanoate at 1 μM, did not inhibit this response. The site of this response was further investigated by examining the effect of PMA on AIF₄^?-induced IP accumulation in canine TSMCs. AIF₄^?-induced IP accumulation was inhibited by PMA treatment, suggesting that G protein(s) can be directly activated by AIF₄^?, which was uncoupled to phospholipase C by PMA treatment. These data conclude that ET-stimulated PI hydrolysis and tracheal smooth muscle contraction are mediated by the activation of ET_Areceptors coupling to a G protein and dependent on the external Ca²⁺. The transduction mechanism of ETs is sensitive to feedback regulation by PKC.     

Immortalized Schwann Cells Express Endothelin Receptors Coupled to Adenylyl Cyclase and Phospholipase C

Endothelins (ETs) are potent regulators of renal, cardiovascular and endocrine functions and act as neurotransmitters in the CNS. Here we report that immortalized Schwann cells express receptors for ETs and characterize some of the cellular events triggered by their activation. Specific binding of [¹²⁵I]-ET-1 to Schwann cell membranes was inhibited by ET-1 and the ET_B-selective agonists ET-3, sarafotoxin 6c and [A1a^1,3,11,15]-ET-1 with IC_50cor values ranging between 2 and 20 nM. No competition was observed with the ET_A receptor-selective antagonist BQ123. Incubation of [³H]-inositol pre-labeled Schwann cells with ET-1, ET-3 or sarafotoxin 6c elicited a concentration-dependent increase in the release of IP₁ that reached a plateau at approximately 100 nM. The efficacy of [Ala^1,3,11,15]-ET-1 (a linear peptide analog of ET-1) was half of that corresponding to ET-1. These stimulatory effects were partially blocked by pre-incubation with pertussis toxin. When Schwann cells were incubated in the presence of 100 nM ET-1 or ET-3 there was a significant inhibition of basal and isoproterenol-stimulated cAMP levels. The inhibitory effects of sarafotoxin 6c and [Ala^1,3,11,15]-ET-1 on isoproterenol-stimulated cAMP levels were similar to that observed with ET-1. Pre-incubation with pertussis toxin completely prevented this effect. These observations indicate that immortalized Schwann cells express receptors for ET peptides (predominantly ET_B) coupled to modulation of phospholipase C and adenylyl cyclase activities. The actions of ETs on Schwann cells provide a novel example of the influence of vascular factors on nerve function.     

Endothelin-1 Binding to Endothelin Receptors in the Rat Anterior Pituitary Gland: Interaction in the Recognition of Endothelin-1 Between ETA and ETB Receptors

1. ¹²⁵I-Endothelin (ET)-1 binding to the rat anterior pituitary gland was saturable and single, with a K _d of 71 pM and a B _max of 120 fmol/mg.2. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, the binding parameters were 8.3 pM and 8.0 fmol/mg, whereas 10 nM sarafotoxin S6c (ET_Bagonist) exerted little change in these binding parameters (K _d,72pM;B _max, 110 fmol/mg).3. ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620, and BQ-788 competitively inhibited ¹²⁵I-ET-1 binding, only when 1.0 M BQ-123 was present in the incubation buffer.4. Thus, the ET_B receptor is capable of binding ET-1 when the ET_A receptor is being occupied by BQ-123. A collaboration mechanism between the ET_A and the ET_B receptor may function in the recognition of ET-1, a typical bivalent ligand.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

Regulatory role of cyclic adenosine 3′,5′-monophosphate on the plattelet cyclooxygenase and platelet function

Thromboxane A₂ plays and important role in arachidonic acid- and prostaglandin H₂-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I₂, prostaglandin I₁, and prostaglandin E₁) and dibutyryl cyclic AMP inhibit both thromboxane A₂ formation and arachidonate-induced aggregation platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A₂ is still formed. Prostaglandin H₂-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A₂ production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cycyooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate mode for studying relationship between the platelet cyclooxygenase and platelet function.     

NLRP3 activation contributes to endothelin-1-induced erectile dysfunction

In the present study, we hypothesized that endothelin (ET) receptors (ET_A and ET_B) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3^−/− and caspase^−/− mice in the presence of ET-1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET-1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET-1 effect. ET-1 decreased CC ACh-, sodium nitroprusside (SNP)-induced relaxation, and increased caspase-1 expression. BQ123 an ET_A receptor antagonist reversed the effect. The ET_B receptor antagonist BQ788 also reversed ET-1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET-1-induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase-1 expression, while BQ788 increased caspase-1 and IL-1β levels in a concentration-dependent manner (100 nM–10 μM). Furthermore, tiron and BAPTA AM prevented ET-1-induced increase in caspase-1. In addition, BAPTA AM blocked ET-1-induced ROS generation. In conclusion, ET-1-induced erectile dysfunction depends on ET_A- and ET_B-mediated activation of NLRP3 in mouse CC via Ca²⁺-dependent ROS generation.     

Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP₃) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca²⁺ ([Ca²⁺]_i) with an EC₅₀ value of 35 µM. In addition, carbachol produced a 1.3–3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E₂ (PGE₂). The M₃ antagonist p-fluorohexahydrosiladifenidol (IC₅₀ = 0.5–0.8 µM) inhibited the increases in [Ca²⁺]_i, IP₃, and cAMP more effectively than the M₁ antagonist pirenzepine (IC₅₀ = 5–9 µM) and the M₂ antagonist methoctramine (IC₅₀ = 20–30 µM). The involvements of [Ca²⁺]_i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 µM) inhibited significantly the synergistic effects of carbachol and PGE₂ on the production of cAMP, whereas the Ca²⁺ ionophore ionomycin (1 µM) clearly enhanced PGE₂-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE₂-stimulated cAMP production. These data suggest that phospholipase C-linked M₃ receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca²⁺-dependent system in the neuronal cells.     

Involvement of phospholipase D activation in endothelin-1-induced release of arachidonic acid in osteoblast-like cells

In a previous study, we have shown that endothelin-1 (ET-1) activates phospholipase D independently from protein kinase C in osteoblast-like MC3T3-E1 cells. It is well recognized that phosphatidylycholine hydrolysis by phospholipase D generates phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol. In the present study, we investigated the role of phospholipase D activation in ET-1-induced arachidonic acid release and prostaglandin E₂ (PGE₂) synthesis in osteoblast-like MC3T3-E1 cells. ET-1 stimulated arachidonic acid release dose-dependently in the range between 0.1 nM and 0.1 μM. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ET-1-induced arachidonic acid release in a dose-dependent manner as well as the ET-1-induced diacylglycerol formation. 1,6-bis-(cyclohexyloxyminocarbonylamino)-hexane (RHC-80267), an inhibitor of diacylglycerol lipase, significantly suppressed the ET-1-induced arachidonic acid release. The pretreatment with propranolol and RHC-80267 also inhibited the ET-1-induced PGE₂ synthesis. These results strongly suggest that phosphatidylcholine hydrolysis by phospholipase D is involved in the arachidonic acid release induced by ET-1 in osteoblast-like cells. J. Cell. Biochem. 64:376–381. © 1997 Wiley-Liss, Inc.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E₂ and 6-keto-prostaglandin F_1α. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 μg/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 μg/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolyses are independently regulated.     

Ca(2+) channels activated by endothelin-1 in CHO cells expressing endothelin-A or endothelin-B receptors

We comparedthe Ca²⁺ channels activated by endothelin-1 (ET-1) inChinese hamster ovary (CHO) cells stably expressing endothelin type A(ET_A) or endothelin type B (ET_B) receptorsusing the Ca²⁺ channel blockers LOE-908 and SK&F-96365. Inboth CHO-ET_A and CHO-ET_B, ET-1 at 0.1 nMactivated the Ca²⁺-permeable nonselective cation channel-1(NSCC-1), which was sensitive to LOE-908 and resistant to SK&F-96365.ET-1 at 1 nM activated NSCC-2 in addition to NSCC-1; NSCC-2 wassensitive to both LOE-908 and SK&F-96365. ET-1 at 10 nM activated thesame channels as 1 nM ET-1 in both cell types, but inCHO-ET_A, it additionally activated the store-operatedCa²⁺ channel (SOCC), which was resistant to LOE-908 andsensitive to SK&F-96365. Up to 1 nM ET-1, the level of the formation of inositol phosphates (IPs) was low and similar in both cell types, but,at 10 nM ET-1, it was far greater in CHO-ET_A than inCHO-ET_B. These results show that, in CHO-ET_Aand CHO-ET_B, ET-1 up to 10 nM activated the sameCa²⁺ entry channels: 0.1 nM ET-1 activated NSCC-1, andET-1  1 nM activated NSCC-1 and NSCC-2. Notably, inCHO-ET_A, 10 nM ET-1 activated SOCCs because of the higherformation of IPs.

     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca2+ signaling

Astrocytes represent a major target for endothelins (ETs), afamily of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in thebrain. In the present study we have investigated the involvement ofdifferent ET receptor subtypes on intercellular dye diffusion, intracellular Ca²⁺homeostasis, and intercellularCa²⁺ signaling in cultured ratastrocytes from hippocampus and striatum. Depending on the ETconcentration and the receptor involved, ET-1- and ET-3-inducedintracellular Ca²⁺ increases withdifferent response patterns. Both ET-1 and ET-3 are powerful inhibitorsof gap junctional permeability and intercellular Ca²⁺ signaling. The nonselectiveET receptor agonist sarafotoxin S6b and theET_B receptor-selective agonist IRL1620 mimicked these inhibitions. The ET-3 effects were only marginallyaffected by an ET_A receptorantagonist but completely blocked by anET_B receptor antagonist. However,the ET-1-induced inhibition of gap junctional dye transfer andintercellular Ca²⁺ signaling wasonly marginally blocked by ET_A orET_B receptor-selective antagonistsbut fully prevented when these antagonists were applied together. TheET-induced inhibition of gap junction permeability and intercellularCa²⁺ signaling indicates thatimportant changes in the function of astroglial communication mightoccur when the level of ETs in the brain is increased.

     

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells

Phospholipase A₂ activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A₂ activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-³H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7–14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E₁ did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A₂ activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A₂, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A₂ activity was also related to previously found differences in the ability of the clones to develop desensitization to β-adrenergic hormones or prostaglandin E₁.     

Effect of microtubule-disrupting agents on superoxide production in human polymorphonuclear leukocytes

We explored the effects of compounds known or proposed to affect microtubule functions on superoxide (O₂⁻) production in human polymorphonuclear leukocytes stimulated by N-formyl-methionyl-phenylalanine (f-Met-Phe), calcium ionophore A23187 and phorbol myristate acetate. F-Met-Phe-induced O₂⁻ production was markedly potentiated not only by microtubule-disrupting agents, including colchicine, vincristine, vinblastine, nocodazole, podophyllotoxin and griseofulvin, but also deuterium oxide (²H₂O), which is proposed to stabilize microtubules, and not affected by lumicolchicine. Ionophore A23187-induced O₂⁻ production was not influenced by colchicine, and markedly enhanced by ²H₂O, whereas phorbol myristate acetate-induced O₂⁻ production was not influenced by colchicine, and slightly inhibited by ²H₂O. ²H₂O did not counteract the effects of colchicine and vice versa. Dibutyryl cyclic AMP and prostaglandin E₁ inhibited O₂⁻ production stimulated by f-Met-Phe and ionophore A23187, whereas phorbol myristate acetate-induced O₂⁻ production was strongly resistant to the inhibitory effect of these agents. The enhancing effect of colchicine and ²H₂O on f-Met-Phe-induced O₂⁻ production was abolished by dibutyryl cyclic AMP. Colchicine promoted concanavalin A cap formation, and ²H₂O produced cancanavalin A patch formation, whereas dibutyryl cyclic AMP did not affect the distribution of concanavalin A receptors. In addition, ²H₂O and dibutyryl cyclic AMP did not interfere with the colchicine-induced concanavalin A cap formation. These findings suggest that f-Met-Phe, ionophore A23187 and phorbol myristate acetate may activate the oxidative metabolism of human polymorphonuclear leukocytes through different mechanisms, and that microtubule-disrupting agents, ²H₂O and cyclic AMP agonists may affect the different steps of the activating system of NAD(P)H oxidase.     

Stimulation of Phospholipase A2 and Secretion of Catecholamines from Brain Synaptosomes by Potassium and A23187

Abstract: Potassium depolarization of rat brain synaptosomes (containing incorporated l-acyl-2-[¹⁴C]arachidonyl-phosphatidylcholine) stimulated endogenous phospholipase A₁ (EC 3.1.1.32) and A₂ (EC 3.1.1.4), as determined by the formation of [¹⁴C]lysophosphatidylcholine, [¹⁴C]arachidonate, and [¹⁴C]prostaglandins, and also stimulated the secretion of [³H]catecholamines. The phospholipase A₂ stimulation, dependent on calcium, was elicited in resting synaptosomes by A23187 and was demonstrated with incorporated 1-acyl-2-[^l4C]oleoyl-phosphatidylcholine but not with incorporated [^I4C]phosphatidylethanolamine or [^l4C]phosphatidylserine. Inhibitors of phospholipase A₂ [p-bromophenacylbromide (10 μM), trifluoperazine (3 μM), and quinacrine (3 μM) reduced the potassium-stimulated [³H]catecholamine release from synaptosomes to 78, 39. and 55%, respectively, of depolarized controls. The addition of lysophosphatidylcholine increased the release of [³H]norepinephrine to levels observed with potassium depolarization, whereas lysophosphatidylethanolamine, lysophosphatidylserine, and sodium dodecyl sulfate were much less effective. Potassium stimulation of synaptosomes increased the endogenous levels of free arachidonic acid and prostaglandins E₂ and F_2α. Indomethacin and aspirin decreased the amounts of prostaglandins formed, allowed the accumulation of free arachidonic acid, and diminished the potassium-stimulated release of [³H]dopamine. p-Bromophenacylbromide inhibited the formation of prostaglandin F_2α. Addition of prostaglandin E₂ inhibited, whereas prostaglandin F_2α enhanced the release of [³H]norepinephrine. These results suggest that calcium influx induced by synaptosomal depolarization activates endogenous phospholipase A₂, with subsequent formation of lysophosphatidylcholine and prostaglandins, both of which may modulate neurosecretion.     

A comparison of responses of guinea-pig isolated trachea to six prostaglandins

Effects of prostaglandins (PG) E₁, E₂, F_2α, A₁, A₂ nad B₂ were studied on guinea-pig isolated tracheal chains. PGF_2α, B₂ and A₂ produced contraction, PGE₁ and E₂ relaxation of the chain, but A₁ produced no response. 1) From the cumulative dose response curves, PGF_2α was more active in producing concentration than B₂ or A₂, though its effect was less than that of acetylcholine (ACh). PGE₁-induced relaxation was less than the response to isoproterenol. 2) PGE₁ and E₂ 1 μg/ml caused a 26.1 ± 3.83% (n=5) or a 9.5 ± 3.36 (n=6) decrease of ACh (1 μg/ml)-induced contraction respectively. The degree of relaxation produced by E₁ was greater than that by E₂ (P<0.01). 3) After five minutes preincubation with each of PGA₁, A₂, B₂ and F_2α in concentrations which did not produce any effect, ACh-induced contraction was augmented only after PGA₂ (P<0.05).     

Characterization and localization of prostaglandin E1 receptors in rat liver plasma membranes

Methodology for measurement and characterization of prostaglandin binding to membranes has been developed. The binding assay was used to study the presence of prostaglandin receptors in high purified cell fractions derived from rat liver. High affinity binding receptors which have a saturation value of 1.0 pmole/mg protein and a dissociation constant of 1.2 nM were found exclusively in the plasma membrane. High affinity receptors were not found in cell fractions containing nuclei, rough microsomes. Golgi complex or mitochondria. The binding by other prostaglandins was competitive with prostaglandin E₁. Competitive binding studies were used to obtain dissociation constants for prostaglandins F_1α, F_2α, B₁, B₂, A₁, A₂, and 15-keto prostaglandin E₂ which were 1100, 100, 300, 180, 16. 16 and 700 nM, respectively. Eicosa-5.8.11.19-tetraynoic acid, an inhibitor of prostaglandin synthesis did not bind appreciably to the prostaglandin E receptor, whereas two prostaglandin analogues, which have high physiological activity compete effectively with prostaglandin E₁ for the receptor. Thus, the binding receptor for the E-type prostaglandins is highly specific both with respect to cell localization as well as the type of substrate. Numerical routines for the fitting of the data and a procedure for the determination of the specific activity of the labelled prostaglandin are provided.     

The effect of acute hypoxia on prostaglandin release in perfused human fetal placenta

The release of prostaglandin E₂ and F_2α, thromboxane B₂ and 6-keto-prostaglandin F_1α was measured in isolated human placental cotyledons perfused under high- and low-oxygen conditions. Also the effect of reoxygenation on prostaglandin production was studied. During the high-oxygen period, prostaglandin E₂ accounted for 44 % and 6-keto-prostaglandin F_1α for 28 % of all prostaglandin release, and the rank order of prostaglandin release was E₂ > 6-keto-prostaglandin F_1α > thromboxane B₂ > prostaglandin F_2α. Hypoxia had no significant effect on quantitative prostaglandin release, but the ration of prostaglandin E₂ to prostaglandin F_2α was significantly increased. After the hypoxic period during reoxygenation the release of 6-keto-prostaglandin F_1α was significantly decreased, as was the ratio of 6-keto-prostaglandin F_1α to thromboxane B₂. Also the ratio of the vasodilating prostaglandins (E₂, 6-keto-prostaglandin F_1α) to the vasocontricting prostaglandins (thromboxane B₂, prostaglandin F_2α) was decreased during reoxygenation period. With the constant flow rate, the perfusion pressure increased during hypoxia in six and was unchanged in three preparation. The results indicate that changes in the tissue oxygenation in the placenta affect prostaglandin release in the fetal placental circulation. This may also have circulatory consequences.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.