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PubMed1993

Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity.

Peptide hormones, neurotransmitters, and autacoids activate a family of seven-transmembrane-domain receptors. Each of these receptors specifically couples to one of several G proteins, Gs, Gi, G(o) and Gp, to activate a specific second messenger system. Cell surface receptors for prostanoids have been characterized pharmacologically and the complementary DNAs for thromboxane A2 receptor and the EP3 subtype of the prostaglandin (PG)E receptor reveal that they belong to the seven-transmembrane-domain receptor family. The EP3 receptor mediates the diverse physiological actions of PGE2 (ref. 3). Although most of them occur through coupling of the EP3 receptor to Gi and inhibition of adenylyl cyclase, the EP3-mediated contraction of uterine muscle can only occur by activation of another second messenger pathway. In chromaffin cells, two different second messenger pathways are activated by PGE2 binding to an apparently single EP3 receptor class. Here we show that at least four isoforms of the EP3 receptor, which differ only at their C-terminal tails and are produced by alternative splicing, couple to different G proteins to activate different second messenger systems.

Adrenal Medulla↗
PubMed1991

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2.

Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration [( Ca2+]i) in cultured LLC-PK1 cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10(-7) M) increased [Ca2+]i from 148 +/- 16 to 288 +/- 39 nM and from 130 +/- 8 to 240 +/- 18 nM, with the values of EC50 for PAF and ONO11113 being 17 +/- 4 and 17 +/- 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO11113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(beta-aminomethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Role of alpha 1-proteinase inhibitor in restraining peritoneal inflammation in CAPD patients.

The concentration and functional state of alpha 1-proteinase inhibitor (alpha 1-PI) may modulate the expression of peritoneal phlogosis by affecting the activity of proteases and synthesis of autacoids. alpha 1-PI is detectable in peritoneal effluents of peritonitis-free patients. alpha 1-PI purified from peritoneal fluid of these patients was biologically active both in terms of inhibition of elastase activity and of synthesis of platelet activating factor (PAF). The biological activity of alpha 1-PI could therefore explain the absence of detectable amounts of PAF in peritonitis free patients despite the presence of intraperitoneal concentrations of tumor necrosis factor-alpha (TNF alpha) that would be sufficient per se to induce the synthesis of PAF. In patients with acute infectious peritonitis, the concentration of immunoreactive alpha 1-PI was significantly increased in respect ot stable patients. However, alpha 1-PI purified from patients with acute peritonitis was functionally inactive both on proteolytic activity on elastase and on TNF alpha-induced PAF synthesis by purified human PMN. The loss of alpha 1-PI activity correlated with the number of peritoneal leukocytes and was probably dependent on oxidative inactivation. Indeed, treatment with reducing agent restored the inhibitory function of alpha 1-PI. The inactivation of alpha 1-PI in patients with peritonitis was associated with the presence of PAF in peritoneal dialysates. These results suggest that alpha 1-PI prevents the proteolytic action and cell activation leading to PAF synthesis in peritonitis free patients. However, inactivation of its function by oxidants generated during the inflammatory process may lead to proteolytic injury and unrestrained synthesis of inflammatory mediators during peritonitis.

Chromatography, High Pressure Liquid↗
PubMed1992

Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids.

A variety of experimental approaches have shown that AVP and mineralocorticoids stimulate Na+ transport through their effects on the number and kinetic properties of amiloride-sensitive Na+ channels in the apical membrane. The different mechanisms by which AVP and mineralocorticoid act on the Na+ channel provide a basis for synergism in their actions, perhaps by a scheme such as that proposed in Figure 5. However, the details of this interaction will require a better understanding of the molecular details involved in activating quiescent channels, increasing their open probability, and reorientating or inserting channels to an operational position in the apical membrane. Electrophysiological and biochemical approaches have gone a long way toward elucidating some of these molecular details. But the latter approach in particular has indicated that the Na+ channel may have multiple regulatory subunits and thus be a target for several intracellular second messengers and autacoids other than those involved in the actions of AVP and aldosterone. The challenges for future research in this area are multiple. It seems likely that the primary amino acid sequence of the channel subunits will soon become available from cloning and sequencing approaches, but the application of this knowledge to understanding how the subunits are integrated into the complete protein and mediate regulatory signals will be a formidable task. It will be important to determine the normal extracellular signals (other than aldosterone and AVP) and the associated intracellular second messengers that alter channel activity. It will also be important to understand how some species such as the rabbit may "turn off" the stimulatory effect of AVP on Na+ reabsorption in the CCD, and how this regulatory process is altered when these cells are cultured. At the whole animal level, it will also be important to investigate whether changes in one or more of the normal regulatory pathways that impinge on the Na+ channel might be involved in a diminished ability to excrete a salt load, as is observed in some models of hypertension. All of these issues need to be understood at the molecular level, and it seems likely they will provide exciting physiological insights at all levels.

Aldosterone↗
PubMed1993

Clonidine, but not bradykinin or ANP, inhibits Na+ and water transport in Dahl SS rat CCD.

We examined the actions of potentially natriuretic autacoids in the isolated perfused cortical collecting duct (CCD) dissected from inbred Dahl (Rapp strain) salt-sensitive rats (SS). Atrial natriuretic peptide (ANP, 10 nM), bradykinin (BK, 10 nM), and clonidine (1 microM) were studied to determine their effects on the lumen-to-bath flux of 22Na+ (J1-->b, pmol min-1 mm-1), hydraulic conductivity (Pf, micron/s), and transepithelial voltage (VT, mV). ANP and BK have been shown by others to significantly reduce net Na+ reabsorption and hydraulic conductivity in the Sprague-Dawley (SD) rat CCD, but previous results from our laboratory showed no ANP or BK effect in the SD CCD. In the present study, we were also unable to observe any effect of either ANP or BK in the SS rat CCD. However, in the presence of AVP, clonidine (a partial alpha 2-adrenergic receptor agonist) significantly reduced J1-->b and Pf from 139 +/- 6 (SEM) to 88 +/- 7 and from 959 +/- 176 to 490 +/- 73, respectively. In addition, clonidine significantly depolarized VT from -14.5 +/- 2.8 to -11.2 +/- 1.8. However, unlike its effects in the SD rat CCD, yohimbine (300 nM, an alpha 2-adrenergic receptor antagonist) did not significantly reverse the effects of clonidine on J1-->b, Pf or VT in the SS rat CCD.

Animals↗
PubMed1997

Mechanism of actions of sumatriptan on coronary flow before and after endothelial dysfunction in guinea-pig isolated heart.

1. The mechanism of action of sumatriptan on coronary flow was examined before and after two different forms of endothelial ablation in guinea-pig isolated hearts. The mechanism was assessed in terms of the influence of the integrity of the coronary endothelium, the role of release of nitric oxide (NO) from the endothelium, and the receptor subtypes mediating the effects. 2. Continuous perfusion with sumatriptan reduced coronary flow, but the concentration-response curve was v-shaped. Sumatriptan (0.001-0.1 microM) caused a concentration-dependent decrease in coronary flow with the maximum effect achieved at 0.23 +/- 0.04 microM. The pEC50 was 8.49 +/- 0.07. At higher concentrations (0.1-10 microM) there was a concentration-dependent diminution of the vasoconstrictor effect. Endothelial ablation by saponin removed the diminution in the vasoconstrictor effect. In contrast pretreatment with NG-nitro L-arginine methyl ester (L-NAME) (100 microM. 45 min perfusion) did not affect it. This was despite both saponin and L-NAME being effective in reducing basal release of NO into the coronary effluent (measured by chemiluminescence) to the same extent (71 +/- 3 and 73 +/- 2%, respectively). 3. GR127935, a selective 5-hydroxytryptamine1D (5-HT1D) receptor antagonist (3 and 10 nM), which by itself had no effect on coronary flow or NO release, antagonized the vasoconstrictor response to sumatriptan and unmasked a sumatriptan-induced concentration-dependent increase in coronary flow and NO release. These increases in coronary flow and NO release were abolished by pretreatment with either saponin or L-NAME. 4. Mesulergine, a 5-HT2 receptor antagonist which had no effect by itself on basal coronary flow or NO release, inhibited the vasodilator response to sumatriptan that occurred in the presence of GR127935, and actually enhanced the vasoconstrictor response, increasing the max mum fall in coronary flow from -3.9 +/- 0.4 to -5.2 +/- 0.4 ml min-1 g-1 (P < 0.05). The diminution of vasoconstrictor effect of sumatriptan was abolished by mesulergine and by pretreatment with saponin, but not by L-NAME. 5. In conclusion, guinea-pig coronary arteries constrict to low concentrations of sumatriptan, causing a reduction in coronary flow. This effect appears to be caused by 5-HT1D agonism with the receptors located on the coronary vascular smooth muscle. With higher concentrations of sumatriptan this is partially offset by a weaker vasodilator effect, which is caused by low affinity 5-HT2 agonism. Although this effect is endothelium-dependent. It is not caused by the release of NO. Interestingly, when the vasoconstrictor effect of sumatriptan was inhibited by the 5-HT1D antagonist GR127935, a high affinity vasodilator effect of sumatriptan was unmasked. This is 5-HT2 receptor mediated and is caused by release of NO from the coronary endothelium. 6. In man, sumatriptan, and 5-HT may both be capable of causing pathogenic coronary vasoconstriction. The implications of the present data are that the scope for this may depend greatly on (i) the extent of underlying endothelial dysfunction, (ii) the extent of endothelial 5-HT2 receptor-mediated release of vasodilator autacoids (which include NO) and (iii) the extent of smooth muscle 5-HT1D receptor-mediated vasoconstriction.

Animals↗
PubMed1997

Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells.

1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca2+ store depletion and Ca2+ entry was studied further. 2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody. 3. Depletion of cellular Ca2+ pools with inositol 1,4,5-trisphosphate (IP3), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca2+ store depletion, chelating of intramicrosomal Ca2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca2+ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP3-sensitive stores. 4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca2+ entry pathways, membrane hyperpolarization due to K+ channel activation or tyrosine kinase activity. 5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca2+/Mn2+ entry to Ca2+ store depletion by 43%, while it did not interfere with intracellular Ca2+ release by IP3 or thapsigargin. 6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca2+/Mn2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9. These data demonstrate that endothelial Cyp450 MO is activated by Ca2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca2+ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca2+ entry activation and increase the driving force for Ca2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

Histamine-induced biphasic macromolecular leakage in the microcirculation of the conscious hamster: evidence for a delayed nitric oxide-dependent leakage.

1. Late effects (up to 3 h) of intravenously-injected histamine on FITC-dextran extravasation were investigated in the conscious hamster, by use of computer-assisted image analysis of fluorescence distribution in a microscopic window of dorsal skin fold preparations. This analysis allowed measurement of local (skin) and general (all organs) extravasations caused by a bolus injection of histamine (1 mg kg(-1), i.v.) 2. Histamine doses higher than 0.01 mg kg(-1) caused biphasic local and general extravasations. Initial phases developed fully within 15 min (for local) and 60 min (for general) and were followed by late phases beginning 90 min after histamine injection. Although the initial and late phases of histamine-induced extravasations had differential apparent reactivities to the autacoid, all the effects of histamine on the microcirculation (1 mg kg[-1]) were inhibited by pyrilamine (1 mg kg(-1), i.v.) but not by cimetidine (1 mg kg(-1), i.v.). 3. Pretreatment with N(G)-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1), i.v.) or N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg kg(-1), i.v.) did not affect the initial phases but did prevent the late phases of local and general extravasations triggered by 1 mg kg(-1) histamine. The inhibitory effects of L-NAME were reversed by L-arginine (30 mg kg[-1]) but not by D-arginine (30 mg kg[-1]) according to the enantioselectivity of nitric oxide synthase (NOS). A late NO-mediated venular dilatation occurred in response to plasma histamine. 4. A low dose of aminoguanidine (1 mg kg(-1), i.v.), a selective inhibitor of the inducible isoform of NOS (iNOS), mimicked the inhibitory effects of L-NAME on the late phases of histamine-induced macromolecular extravasations and venular dilatation. 5. Pretreatment with dexamethasone (1 mg kg(-1), i.v.) prevented both the initial and late phases of histamine-induced extravasations. Fucoidan (1 or 25 mg kg(-1), i.v.) prevented the late phases without affecting initial phases, consistent with a role for leukocytes adhesion in the development of the late NO-mediated effects of histamine. 6. We conclude that intravenous injection of histamine triggers a biphasic inflammatory cascade via initial activation of H1 receptors which induces a late NO-mediated PMN-dependent extravasation process.

Animals↗
PubMed1998

Modulation of airway hyperresponsiveness and eosinophilia by selective histamine and 5-HT receptor antagonists in a mouse model of allergic asthma.

1. Since both histamine and 5-hydroxytryptamine (5-HT) can be released by murine mast cells, we investigated the possible role of these autacoids on airway hyperresponsiveness (AHR), eosinophil infiltration and serum-IgE levels in a murine model of allergic asthma. 2. Ovalbumin-sensitized mice were exposed to either ovalbumin (2 mg ml(-1)) or saline aerosols on 8 consecutive days. Starting one day before the challenge, animals were injected i.p. twice a day with a 5-HT-type 1 (5-HT1) or type 2 (5-HT2) receptor antagonist (methiotepine, 1.25 or 2.0 mg kg(-1) and ketanserin, 12 mg kg(-1), respectively) or a histamine-type 1 (H1) or type 2 (H2) receptor antagonist (mepyramine, 12 or 20 mg kg(-1) and cimetidine, 10 or 25 mg kg(-1), respectively). Furthermore, animals were injected with a combination of cimetidine and ketanserin or with an alpha-adrenoceptor antagonist (phentolamine, 5 mg kg(-1)). 3. In vehicle-treated ovalbumin-challenged animals airway responsiveness to intravenous injections of methacholine in vivo was significantly (9 fold increase, P<0.01) increased when compared to vehicle-treated saline-challenged animals. Furthermore, ovalbumin challenge of vehicle-treated animals induced a significant increase in both eosinophil numbers in bronchoalveolar lavage (BAL) fluid (0+/-0, vehicle/saline and 15.0+/-5.9 x 10(4) cells vehicle/ovalbumin, P<0.05) and ovalbumin-specific IgE levels in serum (157+/-69 and 617+/-171 units ml(-1), respectively, P<0.05) compared to saline-challenged mice. Virtually no eosinophils could be detected in saline-challenged animals after all different treatments. 4. Treatment with ketanserin or cimetidine resulted in a partial but significant decrease of the ovalbumin-induced AHR compared to ovalbumin-challenged controls (P<0.05) and reduced eosinophil infiltration after ovalbumin challenge by 60% and 58%, respectively. The combination of cimetidine and ketanserin almost completely abolished AHR whereas eosinophilia was decreased by 49%. No effects of these antagonists were observed on IL-16 levels in BAL fluid or on serum antigen-specific IgE levels. Treatment with either the H1-receptor, the 5-HT1-receptor or the alpha-adrenoceptor antagonist, did not decrease the observed ovalbumin-induced airway responsiveness or eosinophilia in vehicle-treated animals. Higher doses of either methiotepine (2.0 mg kg(-1)) or mepyramine (20 mg kg(-1)) did decrease ovalbumin-induced eosinophil infiltration (by 67%, P<0.05 and 73%, respectively), whereas no effects of these antagonists were observed on ovalbumin-specific IgE levels in serum. 5. From these data it can be concluded that both histamine and 5-HT play a role in antigen-induced AHR and eosinophilia in the mouse.

Airway Resistance↗
PubMed2001

Role of ETB and B2 receptors in the ex vivo platelet inhibitory properties of endothelin and bradykinin in the mouse.

1. We have developed a model to study the inhibitory properties of endogenous autacoids triggered by systemically-administered vasoactive peptides, on platelet aggregation ex vivo in the mouse. 2. Adenosine diphosphate (ADP) (0.5-10 microM) induces a concentration-dependent aggregation of platelet-rich plasma derived from C57BL/6 mice. Intravenously-administered endothelin-1 (0.01-1 nmolx kg(-1)), the selective ETB agonist, IRL-1620 (0.0 -1 nmol x kg(-1)) or bradykinin ( 1-100 nmol x kg(-1)) significantly reduced in a dose-dependent fashion the ADP-induced platelet aggregation. 3. The non-selective cyclo-oxygenase (COX) inhibitor, indomethacin, a selective COX-2 inhibitor NS-398 or the prostacyclin synthase inhibitor, tranylcypromine (10 mg x kg(-1)), markedly reduced the inhibitory properties of endothelin-1, whereas only a combination of both indomethacin, NS-398 or tranylcypromine and L-NAME (10 mg x kg(-1)) were required to abolish the response to bradykinin. 4. An ETB-selective antagonist (BQ-788) or knockout of the B2 receptor gene (in B2 knockout mice) abolishes the platelet inhibitory properties of endothelin-1 and bradykinin, respectively. 5. Our results suggest that intravenously-administered endothelin-1 and bradykinin, through ETB and B2 receptor activation, respectively, inhibit platelet aggregation ex vivo in the mouse. The inhibitory properties of endothelin-1 require the activation of COX-2 and the subsequent generation of prostacyclin. In addition to the two previously mentioned factors, nitric oxide is required for the anti-aggregatory effects of bradykinin.

Adenosine Diphosphate↗
PubMed2004

Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP.

1. Histamine is generally regarded as a pro-inflammatory mediator in diseases such as allergy and asthma. A growing number of studies, however, suggest that this autacoid is also involved in the downregulation of human polymorphonuclear leukocyte (PMN) functions and inflammatory responses through activation of the Gs-coupled histamine H(2) receptor. 2. We report here that histamine inhibits thapsigargin- and ligand (PAF and fMLP)-induced leukotriene (LT) biosynthesis in human PMN in a dose-dependent manner. 3. The suppressive effect of histamine on LT biosynthesis was abrogated by the histamine H(2) receptor antagonists cimetidine, ranitidine, and tiotidine. In contrast, the histamine H(1), H(3), and H(4) receptor antagonists used in this study were ineffective in counteracting the inhibitory effect of histamine on the biosynthesis of LT in activated human PMN. 4. The inhibition of LT biosynthesis by histamine was characterized by decreased arachidonic acid release and 5-lipoxygenase translocation to the nuclear membrane. 5. Incubation of PMN with the cAMP-dependent protein kinase (PKA) inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide prevented the inhibitory effect of histamine on LT biosynthesis, suggesting an important role for PKA in this effect of histamine on LT biosynthesis in PMN. 6. These data provide the first evidences that, similarly to adenosine and prostaglandin E(2), histamine is a potent suppressor of LT biosynthesis, and support the concept that histamine may play a dual role in the regulation of inflammation.

Arachidonate 5-Lipoxygenase↗
PubMed2000

Role of bradykinins and nitric oxide in the AT2 receptor-mediated hypotension.

Footshocks increases mean arterial pressure and heart rate. Systemic or intracerebroventricular (IVT) administration of losartan, a specific angiotensin AT1 receptor antagonist, not only inhibited the pressor response to footshocks but resulted in vasodepression. Peripheral or IVT administration of PD 123319, a specific angiotensin AT2 receptor antagonist, did not alter the haemodynamic response to footshocks. However, simultaneous blockade of angiotensin AT1 and AT2 receptors by combined systemic or central administration of losartan and PD 12319, eliminated the vasodepressor response to footshocks unmasked in losartan pretreated rats. Our data suggest that activation of peripheral or brain angiotensin AT2 receptor mediated the vasodepressor response to footshocks in the presence of angiotensin AT1 receptor antagonist. We studied the role of kinins and nitric oxide in the vasodepressor response observed after footshocks. The decrease in mean arterial pressure observed after footshocks in losartan treated rats was blunted by systemic or IVT administration of icatibant (HOE 140) or N(G)-nitro-L-arginine-methyl ester, indicating that peripheral or brain kinins and nitric oxide are involved in the hypotensor response to footshocks during angiotensin AT1 receptor blockade. Our results suggest a role for angiotensin AT2 receptor in the regulation of arterial blood pressure, possibly through the release of vasodilator autacoids such as bradykinins and nitric oxide.

Adrenergic beta-Antagonists↗
PubMed1989

Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells.

The metabolism of platelet-activating factor (PAF; identified as AGEPC: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (lyso-GEPC: 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) was investigated in cultured rat Kupffer cells. The rat Kupffer cells accumulated [3H]AGEPC and deacetylated this compound to the corresponding [3H]lyso-GEPC, which was the major metabolic product of [3H]AGEPC. [3H]Lyso-GEPC was distributed primarily in the supernatant fraction of incubated cells throughout the experimental interval. Only a very small portion of the [3H]lyso-GEPC was further converted to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), indicating that this acylation process was not particularly active in these cells. When [3H]lyso-GEPC was incubated with Kupffer cells, the conversion of lyso-GEPC to AGEPC via the acetyltransferase reaction increased up to 30 min and declined thereafter. Bovine serum albumin (BSA) had a substantial influence on both the cellular uptake and the metabolism of [3H]AGEPC. An increase in the BSA concentration in the incubation media reduced the cellular uptake of [3H]AGEPC and the subsequent formation of lyso-GEPC. The results of this study suggest that the hepatic Kupffer cells play an important role in the metabolism of PAF. Moreover, these results infer that the regulation of the PAF level in certain hepatic pathophysiological situations may be a consequence of the production and subsequent metabolism of this potent lipid autacoid in the Kupffer cells of the liver.

Animals↗
PubMed1993

Glycogenolytic and haemodynamic responses to bovine serum albumin in isolated perfused livers from sensitized rats.

Infusion of BSA into isolated perfused livers of rats sensitized by intraperitoneal injection of BSA led to rapid increases in portal-vein pressure, glucose output and the lactate/pyruvate ratio in the effluent perfusate, with concomitant decreases in oxygen consumption and lactate+pyruvate efflux. The responses were attenuated at low (approximately 7 microM) perfusate Ca2+, but were restored on re-addition of normal Ca2+ concentration. Co-infusion of the cyclo-oxygenase inhibitor ibuprofen (50 microM) or of the platelet-activating factor receptor antagonist WEB 2170 (1.2 microM) inhibited haemodynamic responses to BSA (5 micrograms/ml) by 48% and 59% respectively. Responses to BSA were also attenuated by prior infusion of the beta-adrenergic agonist isoprenaline. Glycogen phosphorylase a activity was increased by 26% in livers freeze-clamped 2 min after onset of BSA infusion; tissue prostaglandin E2 content was increased at 2 min, but returned to control levels at 5 min. Homologous desensitization of hepatic responses to BSA was observed, but heterologous desensitization with heat-aggregated IgG did not take place. It is concluded that livers from rats sensitized to antigen respond directly to subsequent antigen administration by vasoconstriction and glycogenolysis, and that autacoid mediators are involved in these responses.

Animals↗
PubMed1996

Action of endothelin-1 on glomerular haemodynamics in the dog: lack of direct effects on glomerular ultrafiltration coefficient.

1. Although numerous studies have demonstrated the potent vasoconstrictor effect of endothelin-1 on the renal vasculature, it is difficult to differentiate in vivo between indirect and direct actions of endothelin. 2. In the present study we therefore performed micropuncture experiments in 14 anaesthetized dogs after the administration of endothelin-1 into the renal artery (i.r.a.) for 15 min at a dose of 2.78 ng min-1 kg-1 which did not affect blood pressure or contralateral kidney function. 3. In seven dogs on a 'normal' sodium diet (3.5 mmol of NaCl day-1 kg-1) endothelin-1 resulted in decreases in renal blood flow and glomerular filtration rate with a rise in filtration fraction from 0.24 +/- 0.01 to 0.35 +/- 0.01. Similar changes were seen at the single-nephron level. An increase from 0.11 +/- 0.01 to 0.23 +/- 0.02 (+109 +/- 11%) in efferent, and from 0.15 +/- 0.01 to 0.24 +/- 0.02 mmHg min-1 microliter-1 (+58 +/- 4%) in afferent arteriolar resistance was observed. The ultrafiltration coefficient, Kf, decreased from 4.75 +/- 0.14 to 3.67 +/- 0.14 microliter min-1 mmHg-1 (P < 0.01). 4. In another group of seven dogs, the same endothelin-1 infusion was given after an administration into the renal artery of a 'cocktail' designed to block the receptors of alpha- and beta-adrenergic agonists, thromboxane A2, leukotrienes and angiotensin II together with intravenous cyclo-oxygenase inhibition. Under these conditions, the vasoconstrictor action of endothelin-1 was reduced by more than 30%. The increase in vascular resistance was now relatively similar in both arterioles, i.e. +44 +/- 5% in the afferent and 66 +/- 6% in the efferent arterioles (no significant difference). No change in Kf was seen after the 'cocktail'-blockade (9.4 +/- 0.4 versus 10.1 +/- 0.1 microliter min-1 mmHg-1). 5. In conclusion, in anaesthetized dogs on a 'normal' salt diet, the vasoconstrictor effect of endothelin-1 is more pronounced on the efferent than on the afferent arteriole. This is accompanied by decreases in glomerular filtration rate and water, urea and electrolyte excretion. Part of the vasoconstrictor action of endothelin seems to be mediated by other autacoids. Endothelin-1 itself contributes only partly to pre- and post-glomerular vasoconstriction and has no direct effect on the glomerular ultrafiltration coefficient Kf.

Adrenergic alpha-Antagonists↗
PubMed1999

Effects of hydrogen peroxide on the guinea-pig tracheobronchial mucosa in vivo.

Lumenal entry of plasma (mucosal exudation) is a key feature of airway inflammation. In airways challenged with histamine-type mediators and allergen the mucosal exudation response occurs without causing epithelial derangement and without increased airway absorption. In contrast, reactive oxygen metabolites may cause mucosal damage. In this study, involving guinea-pig airways, we have examined effects of H2O2 on airway exudation and absorption in vivo. Vehicle or H2O2 (0.1 and 0.5 M) was superfused onto the tracheobronchial mucosal surface through an oro-tracheal catheter. 125I-albumin, given intravenously, was determined in tracheobronchial tissue and in lavage fluids 10 min after challenge as an index of mucosal exudation of plasma. The tracheobronchial mucosa was also examined by scanning electron microscopy. In separate animals, 99mTc-DTPA was superfused 20 min after vehicle or H2O2 (0.1 and 0.5 M) had been given. A gamma camera determined the disappearance rate of 99mTc-DTPA from the airways as an index of airway absorption. The high dose of H2O2 (0.5 M) produced epithelial damage, increased the absorption of 99mTc-DTPA (P < 0.001), and increased the exudation of plasma (P < 0.001). Notably, it appeared that all extravasated plasma had entered the airway lumen within 10 min. These data demonstrate that H2O2 differs from exudative autacoids such as histamine by causing both epithelial damage and plasma exudation responses. These data also agree with the view that the epithelial lining determines the rate of absorption and is responsible for the valve-like function that allows lumenal entry of extravasated bulk plasma without any increased inward perviousness.

Albumins↗
PubMed1996

8-Epi PGF2 alpha: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo.

1. Excessive free radical generation is thought to contribute to tissue injury in a broad spectrum of diseases. A particular constraint in addressing this hypothesis has been the inability to assess free radical generation in vivo and the lack of information on drugs or vitamins which act as effective antioxidants in vivo. 2. Traditional approaches have relied upon measures of substrate oxidizability or spin trapping of free radical adducts ex vivo. It is unknown how these measurements might relate, in a quantitative fashion, to the generation of reactive oxygen species in vivo. Isoeicosanoids are free radical catalyzed products of arachidonic acid. One of these compounds, 8-epi prostaglandin F2 alpha (8-epi PGF2 alpha) exhibits biological activity and may function as an autacoid. Specific analysis of this 8-epi PGF2 alpha isomer indicates that it is elevated in certain syndromes thought to be associated with oxidant stress. These include vascular reperfusion, paracetamol poisoning and liver cirrhosis. Apparently healthy individuals who smoke cigarettes or consume alcohol exhibit dose dependent increments in excretion of 8-epi PGF2 alpha. Excretion is depressed by antioxidant vitamins, although not by the nonspecific cyclooxygenase (COX) inhibitor, aspirin, even though 8-epi PGF2 alpha may be formed by either COX-1 or COX-2. 3. Specific analysis of this and other isoeicosanoids may afford an opportunity to evaluate the effects of antioxidant interventions in human diseases characterized by excessive lipid peroxidation in vivo.

Animals↗
PubMed1998

Biosynthesis and degradation of bioactive fatty acid amides in human breast cancer and rat pheochromocytoma cells--implications for cell proliferation and differentiation.

The endogenous cannabinoid, anandamide (arachidonoylethanolamide), and the sleep-inducing factor, oleamide (cis-9-octadecenoamide), represent two classes of long-chain fatty acid amides with several neuronal actions and metabolic pathways in common. Here we report that these two compounds are present in human breast carcinoma EFM-19 cells and rat adrenal pheochromocytoma PC-12 cells, together with the enzyme responsible for their degradation, fatty acid amide hydrolase, and the proposed biosynthetic precursors for arachidonoylethanolamide and related acylethanolamides, the N-acyl-phosphatidylethanolamines. Lipids extracted from cells labelled with [14C]ethanolamine contained radioactive compounds with the same chromatographic behaviour as arachidonoylethanolamide and acyl-PtdEtns. The levels of these compounds were not influenced by either stimulation with ionomycin in EFM-19 cells or two-week treatment with the nerve growth factor in PC-12 cells. The chemical nature of arachidonoylethanolamide, related acylethanolamides and the corresponding acyl-PtdEtns was confirmed by gas chromatographic/mass spectrometric analyses of the purified compounds, which also showed the presence of higher levels of oleamide. The latter compound, which does not activate the central CB1 cannabinoid receptor, exhibited an anti-proliferative action on EFM-19 cells at higher concentrations than arachidonoylethanolamide (IC50 = 11.3 microM for oleamide and 2.1 microM for arachidonoylethanolamide), while at a low, inactive dose it potentiated an arachidonoylethanolamide cytostatic effect. The CB1 receptor selective antagonist SR 141716A (0.5 microM) reversed the effect of both arachidonoylethanolamide and oleamide. EFM-19 cells and PC-12 cells were found to contain a membrane-bound [14C]arachidonoylethanolamide-hydrolysing activity with pH dependency and sensitivity to inhibitors similar to those previously reported for fatty acid amide hydrolase. This enzyme was inhibited by oleamide in both intact cells and cell-free preparations. The presence of transcripts of fatty acid amide hydrolase in these cells was shown by northern blot analyses of their total RNA. The rate of [14C]arachidonoylethanolamide hydrolysis by intact cells, the kinetic parameters of arachidonoylethanolamide enzymatic hydrolysis and the amounts of the fatty acid amide hydrolase transcript, were not significantly influenced by a two-week treatment with nerve growth factor and subsequent transformation of PC-12 cells into neuron-like cells. These data show for the first time that: (a) induction by nerve growth factor of a sympathetic neuronal phenotype in PC-12 cells has no effect on arachidonoylethanolamide/oleamide metabolism, (b) arachidonoylethanolamide and oleamide are autacoid suppressors of human breast cancer cell proliferation. Moreover these data lend conclusive support to the previous hypothesis that oleamide may act as an enhancer of arachidonoylethanolamide actions through competitive inhibition of its degradation.

Amidohydrolases↗