Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A2

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1-induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1 alpha. ET-1 increased 6-keto-PGF1 alpha formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1 alpha formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF1 alpha production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF1 alpha formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF1 alpha formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.     

Alpha-1A adrenergic receptor stimulation with phenylephrine promotes arachidonic acid release by activation of phospholipase D in rat-1 fibroblasts: inhibition by protein kinase A

This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modulation by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A > alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR-expressing R-1Fs. 8-(4-chlorophenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, alpha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A.     

Alpha adrenergic receptor subtypes involved in prostaglandin synthesis are coupled to Ca++ channels through a pertussis toxin-sensitive guanine nucleotide-binding protein

Previously, we have shown that alpha-2C and alpha-1A adrenergic receptors (AR) stimulate prostacyclin (PGI2) synthesis through a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in vascular smooth muscle cells (VSMC). The purpose of this study was to assess the role of Ca++ in PGI2 production elicited by alpha-AR activation and to investigate the modulation of the Ca++ channel by G proteins coupled to these alpha-AR in VSMC. PGI2 was measured as immunoreactive 6-keto-PGF1 alpha by radioimmunoassay and cytosolic calcium ([Ca++]i) by spectrofluorometry using fura-2. Norepinephrine, methoxamine and UK-14304 enhanced 6-keto-PGF1 alpha production and [Ca++]i, which was inhibited by depletion of extracellular Ca++ and by Ca++ channel antagonists (verapamil, nifedipine and PN 200-110). Moreover, the Ca++ channel activator Bay K 8644 increased 6-keto-PGF1 alpha production in a nifedipine-sensitive manner, indicating the involvement of dihydropyridine-sensitive Ca++ channels in VSMC. Pertussis toxin inhibited AR agonist-induced 6-keto-PGF1 alpha production and the increase in [Ca++]i. Alpha AR agonists increase Ca++ influx in the presence of guanosine 5'-0-(2- thiodiphosphate) (GTP-gamma-S), and this effect was blocked in the presence of guanine 5'-O-(2-thiodiphosphate) (GDP-beta-S) and antiserum against Gi alpha 1-2 protein in reversibly permeabilized cells with beta-escin. VSMC of rabbit aortae contain a G protein(s) that was recognized by Gi alpha 1-2 but not Gi alpha 3 or G0 antibodies at 1:200 dilution. The calmodulin inhibitor W-7 blocked AR agonist and Bay K 8644-stimulated 6-keto-PGF1 alpha production. The phospholipase A2 inhibitors 7,7-dimethyleicosadienoic acid and oleoyloxyethyl phosphocholine but not phospholipase C inhibitor U-73122 reduced 6-keto-PGF1 alpha production in VSMC. These data suggest that a pertussis toxin-sensitive G protein, probably Gi alpha 1-2, coupled to alpha AR regulates Ca++ influx, which, in turn, by interacting with calmodulin, increases phospholipase A2 activity to release arachidonic acid for PGI2 synthesis in VSMC of rabbit aortae.     

Establishment of microanalysis of prostaglandin metabolites by GC/MS and its clinical application

We established microdetermination methods of prostaglandin (PG) metabolites by GC-selected ion monitoring (GC-SIM) and applied them to the clinical investigations. At first the microdetermination of delta 17-6-keto-PGF1 alpha, a hydrolyzed metabolite of PGI2, is described. An authentic delta 17-6-keto-PGF1 alpha was prepared from eicosapentaenoic acid (EPA) incubated with a homogenate from the bovine aortic intima. [18O] delta 17-6-Keto-PGF1 alpha was synthesized to obtain an internal standard for GC-SIM of delta 17-6-keto-PGF1 alpha. A good linear response over the range of 10 pg-5 ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of delta 17-6-keto-PGF1 alpha and 6-keto-PGF1 alpha. Furthermore, a monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-PGF1 alpha in the human blood sera. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in the human blood sera by GC/MS analysis. We were able to detect delta 17-6-keto-PGF1 alpha of the amount ranging from 6 to 26 pg/ml in the human blood plasma. The present method can be applied to the determination of delta 17-6-keto-PGF1 alpha in the human urine and plasma. Diabetes mellitus induces platelet alterations such as hyperaggregation. Variations in PG production seem to be related to this phenomenon but the changes in PG levels remain unclear. So we microanalyzed the 11-dehydrothromboxane B2 (TXB2) and 2,3-dinor-6-keto-PGF1 alpha, which were stable metabolites of TXA2 and PGI2, in the urine and investigated the relationship between the thromboxane/prostacyclin (TX/PGI) ratio and diabetes mellitus. The TX/PGI ratio in the urine of diabetics was higher than that of healthy volunteers. In murine, the TX/PGI ratio of STZ-induced mice was also higher than that of non-induced mice. The ratio of db/db mice also increased with the progress of diabetes mellitus. Furthermore, we investigated the relationship between the retinal vein occlusion (RVO), a thrombotic disease in which the retinal vein is blocked by blood aggregations, and the TX/PGI ratio. The TX/PGI level in patients with the RVO, who were not combine diabetes, was significantly higher than that in healthy volunteers. One of the causes of the RVO may be due to the variation of thromboxane production. This GC-SIM method can be used to determine the TX/PGI ratio in the urine.     

Neurotransmitter-mediated regulatory mechanisms of axoplasmic transport--acetylcholine and adrenaline

It has not been known whether or not the axoplasmic transport depends on regulatory influences mediated by neurotransmitters. The video-enhanced microscope technique has made it possible to visualize the real-time movement of micro-particles along axons and thus to observe its quick response to external signals. Using this technique, the effects of acetylcholine (ACh) and adrenaline on the axoplasmic transport in cultured cervical ganglion (SCG) cells were examined. Application of ACh inhibited the transport in both anterograde and retrograde directions. This effect was mimicked by the muscarinic receptor agonist arecoline, but not by the nicotinic receptor agonist nicotine. The response to ACh was inhibited by QNX (3-quinuclidinyl-xanthine-9-carboxylate), a muscarinic receptor antagonist. Immunocytochemistry and in situ hybridization with anti-muscarinic receptor subtypes compounds demonstrated the expression of m2 receptors on the SCG cell. Islet-activation protein (IAP), a G-protein inhibitor, completely blocked the suppressive effect of ACh. The protein kinase A (PKA) inhibitor KT-5720 induced a similar effect to that of ACh. In contrast to the effect of ACh, adrenaline increased both anterograde and retrograde transport. The beta 2-receptor agonist (albuterol), but not alpha-receptor agonists (phenylphrine and clonidine) or beta 1-receptor agonist (dobutamine), mimicked the effect of adrenaline. The beta 2-receptor antagonist butoxamine abolished the facilitatory response to adrenaline. Dibutyryl cyclic AMP, a membrane permeable cAMP, and forskolin, an activator of adenylyl cyclase, induced a similiar effect to that of adrenaline. These results suggest that 1) ACh, acting through m2-receptors, activates Gi-protein and thus inhibits cAMP synthesis, 2) adrenaline, acting through beta 2-receptors, increases intracellular cAMP concentration, and 3) these changes in cyclic AMP levels inhibit or enhance the activity of PKA to phosphorylate proteins related to the axoplasmic transport.     

Differential coupling of alpha1-, alpha2-, and beta-adrenergic receptors to mitogen-activated protein kinase pathways and differentiation in transfected PC12 cells

Three adrenergic receptor families that selectively activate three different G proteins (alpha1/Gq/11, alpha2/Gi, and beta/Gs) were used to study mitogen-activated protein kinase (MAPK) activation and differentiation in PC12 cells. PC12 cells were stably transfected with alpha1A-, alpha2A-, or beta1-adrenergic receptors (ARs) in an inducible expression vector, and subclones were characterized. Norepinephrine stimulated inositol phosphate formation in alpha1A-transfected cells, inhibited cyclic adenosine 3'5'-monophosphate (cAMP) formation in alpha2A-transfected cells, and stimulated cAMP formation in beta1-transfected cells. Nerve growth factor activated extracellular signal-regulated kinases (ERKs) in all cell lines; however, norepinephrine activated ERKs only in alpha1A- and beta1-transfected cells but not in alpha2A-transfected cells. Norepinephrine also activated c-Jun NH2-terminal kinase and p38 MAPK in alpha1A-transfected cells but not in beta1- or alpha2A-transfected cells. Norepinephrine caused differentiation of PC12 cells expressing alpha1A-ARs but not those expressing beta1- or alpha2A-ARs. However, norepinephrine acted synergistically with nerve growth factor in promoting differentiation of cells expressing beta1-ARs. Whereas ERKs are activated by Gi- but not Gs-linked receptors in many fibroblastic cell lines, we observed the opposite in PC12 cells. The results show that activation of the different G protein signaling pathways has different effects on MAPKs and differentiation in PC12 cells, with Gq signaling pathways activating all three major MAPK pathways.     

Relation of the levels of systemic prostaglandins and hemodynamic changes present during orthotopic transplant of the liver]

The aim of this study is to investigate the role of prostaglandins (PGI2 and TXA2) in relation with the hemodynamic alterations occuring after graft reperfusion in patients undergoing OLT. A total of 40 patients with liver cirrhosis were studied. Systemic 6-keto-PGF1 alpha and TXB2, stable metabolites of PGI2 and TXA2 respectively, were determined at the radial artery, at four different surgical stages: basal, hepatectomy, anhepatic, and 10 minutes after graft reperfusion. Overall results showed that 6-keto-PGF1 alpha levels were significantly elevated during hepatectomy (1143 +/- 204) when compared to values in the basal stage (p = 0.007). During hepatectomy, 6-keto-PGF1 alpha levels did not correlate to systemic vascular resistance index (SVRI), neither with the cardiac index (IC) nor with the medial arterial pressure (MAP) in the same stage. During the anhepatic stage, only IRVS was inversely correlated with 6-keto-PGF1 alpha levels (p = 0.004): there was no relation with MAP and CI. During reperfusion no correlations were observed between 6-keto-PGF1 alpha levels and MAP, CI or SVRI. We conclude that systemic PGI2 levels are very high in cirrhotic patients undergoing OLT. The absence of correlation between the magnitude of changes in hemodynamic parameters and 6-keto-PGF1 alpha levels during reperfusion of the new liver suggests that other factors must play a role in these hemodynamic changes.     

Echinococcus granulosus infection of farm dogs of Iran

The non-enzymatic metabolites of prostacyclin (PGI2) and thromboxane A2 (TXA2), 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and thromboxane B2 (TXB2), and their 2,3-dinor metabolites, 2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-TXB2, were measured in early morning urine samples in 24 in vitro fertilization (IVF) cycles in 24 women and in 27 women who became pregnant after IVF and embryo transfer (ET). The sum of the non-enzymatic metabolites and their 2,3-dinor metabolites was considered to be a reflection of total PGI2 and total TXA2 production in vivo. Both the ratio of 'total' PGI2/'total' TXA2 and the ratio of the 2,3-dinor metabolites were calculated. TXB2 concentrations showed virtually no change and the ratios of the non-enzymatic metabolites of PGI2 and TXA2 versus their 2,3-dinor metabolites remained relatively constant. As a consequence, the ratio of 2,3-dinor-6-keto-PGF1alpha/2,3-dinor-TXB2 was a close reflection of the ratio of 'total' PGI2/'total' TXA2, although the latter ratio was significantly higher all the time. We conclude that for comparative studies on the balance between PGI2 and TXA2 in IVF cycles and during gestation, the determination of the 2,3-dinor metabolites alone can replace the measurement of all four metabolites.     

Cyclo-oxygenase products released by low pH have capsaicin-like actions on sensory nerves in the isolated guinea pig heart

OBJECTIVE: Previous work has shown that ischaemia releases calcitonin gene related peptide (CGRP) from capsaicin sensitive nerve terminals in the perfused heart. Prostacyclin (PGI2) is also released during ischaemia. The aim of this study was to investigate whether the release of CGRP by low pH and lactic acid was associated with PGI2 formation and if PGI2 mediated its effect through capsaicin receptors which could be inhibited by capsazepine. METHODS: The isolated Langendorff perfused guinea pig heart was used with a constant perfusion pressure of 70 cm H2O. Low pH was accomplished by changing the Tyrode solution to buffers with pH 7, 6, and 5, or lactic acid (5 mM with pH 6.9). The outflow of CGRP and the stable PGI2 metabolite 6-keto-PGF1 alpha was measured by radioimmunoassay. RESULTS: Low pH (pH 7, 6, 5) and lactic acid evoked release of CGRP. At moderate acidosis (pH 7 and 6) the CGRP release was dependent on extracellular Ca2+, while at pH 5 approximately half of the peptide release persisted in the absence of extracellular Ca2+. This release was attenuated by diclofenac or indomethacin, two inhibitors of prostaglandin formation, as well as by the capsaicin receptor antagonist capsazepine. Both arachidonic acid and PGI2, the predominant cyclo-oxygenase product formed during myocardial ischaemia, evoked a capsazepine sensitive release of CGRP, while capsazepine did not influence the formation of PGI2 evoked by low pH or arachidonic acid. CONCLUSIONS: In the isolated guinea pig heart, moderate acidosis is associated with CGRP release dependent on influx of extracellular Ca2+ and formation of PGI2, with subsequent stimulation of capsazepine sensitive receptors. With more severe acidosis there is an additional non-PGI2-linked CGRP release. Capsazepine represents a novel pharmacological principle for inhibiting the effects of prostanoids on sensory nerves without influencing their formation.     

Increased production of eicosanoids, TXA2, PGI2 and LTC4 in experimental spinal cord injuries

Arachidonate metabolites have many kinds of bioactivities. Thromboxane A2 (TXA2) stimulates platelet aggregation and vasoconstriction, whereas prostaglandin I2 (PGI2) antagonises its activities. Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) are determined in biological materials. Production of TXB2, 6-keto-PGF1 alpha and leukotriene C4 (LTC4), which have potent vascular permeability, was measured by radioimmunoassay in experimental spinal cord injured animals. TXB2 level in the rat spinal cord reached a peak concentration of 133.6 +/- 3.8 pmol/g cord, and 6-keto-PGF1 alpha increased to 26.2 +/- 11.7 pmol/g cord 5 minutes after the injury. There was good correlation between TXB2 production and vascular damage as monitored by fluorescein uptake. When OKY-046 ((E)-3-[4-(1-imidazolylmethyl) phenyl]-2-propenoic acid), which selectively inhibits TXA2 synthetase activity, was administered 10 minutes before injury, the increase in TXB2 production was inhibited by more than 80%, but the degree of vascular damage was reduced by only 40%. In the guinea pig spinal cord, LTC4 levels reached a peak concentration of 2.2 +/- 0.4 pmol/g cord 10 minutes after compression, while that of TXB2 reached 146.8 +/- 6.2 pmol/g cord. The increased production of TXB2 was correlated with the degree of compression injury while that of LTC4 production did not. These findings suggest that vasoactive eicosanoids, TXA2, PGI2 and LTC4, play important roles in secondary damage following spinal cord injury, although their roles may be different among species of animals.     

Beta 1 integrin cross-linking inhibits CD16-induced phospholipase D and secretory phospholipase A2 activity and granule exocytosis in human NK cells: role of phospholipase D in CD16-triggered degranulation

Recent data indicate that integrin-generated signals can modulate different receptor-stimulated cell functions in both a positive (costimulation) and a negative (inhibition) fashion. Here we investigated the ability of beta 1 integrins, namely alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors, to modulate CD16-triggered phospholipase activation in human NK cells. beta 1 integrin simultaneous cross-linking selectively inhibited CD16-induced phospholipase D (PLD) activation, without affecting either phosphatidylinositol-phospholipase C or cytosolic phospholipase A2 (PLA2) enzymatic activity. CD16-induced secretory PLA2 (sPLA2) protein release as well as its enzymatic activity in both cell-associated and soluble forms were also found to be inhibited upon beta 1 integrin coengagement. The similar effects exerted by specific PLD pharmacological inhibitors (2,3-diphosphoglycerate, ethanol) suggest that in our experimental system, sPLA2 secretion and activation are under the control of a PLD-dependent pathway. By using pharmacological inhibitors (2,3-diphosphoglycerate, wortmannin, ethanol) we also demonstrated that PLD activation is an important step in the CD16-triggered signaling cascade that leads to NK cytotoxic granule exocytosis. Consistent with these findings, fibronectin receptor engagement, by either mAbs or natural ligands, resulted in a selective inhibition of CD16-triggered, but not of PMA/ionomycin-induced, degranulation that was reversed by the exogenous addition of purified PLD from Streptomyces chromofuscus.     

Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA

The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell-free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5'-3-O-(thio)triphosphate) (GTPgammaS), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C2-(N-acetyl-), C8- (N-octanoyl-), and long-chain ceramides, but not dihydro-C2-ceramide, inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cell-free system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C2- and C8-ceramides prevented the GTPgammaS-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C2-ceramide, but not dihydro-C2-ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-alpha and -beta1 that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-epsilon and -zeta. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity.     

Long-term observations of uterine contractions in nonpregnant dogs

Uterine contractile activity in nonpregnant conscious dogs was investigated based on 2- to 6-mo-long continuous recording by means of a chronically implanted force transducer. We found that nonpregnant uterine contractile activity could be classified into six major patterns: sporadic contractions, weak and strong tonic contractions, weak and strong phasic contractions, and phasic contraction bursts. The contractile patterns during proestrus and estrus were the most active, with strong phasic and tonic contractions and phasic contraction bursts. The phasic and tonic contractions were inhibited dose-dependently by a beta 2 adrenergic agonist, ritodrine, and reproduced by an alpha 2 adrenergic agonist, clonidine. In contrast, the cholinergic inhibitors atropine and hexamethonium did not affect the spontaneous occurrence of these contractions, although bethanechol evoked uterine contractions. Oxytocin and prostaglandin F2 alpha-induced contractions were phasic during estrus, whereas they showed tonic increases with phasic contractions during proestrus, diestrus, and anestrus, and these contractions did not resemble the spontaneous contractions. In conclusion, the nonpregnant uterus contracts continuously in harmony with the estrous cycle phases, and its contractile activity is enhanced by alpha adrenergic receptors and inhibited by beta 2 adrenergic receptors.     

Alteration of thromboxane A2/prostacyclin and their effect on spinal cord blood flow in experimental spinal cord injury in rats

In order to document the contribution of Thromboxane (TXA2) and Prostacyclin (PGI2) to the secondary damage following spinal cord injury (SCI) and their effects on spinal cord blood flow (SCBF), the alteration of SCBF, TXB2 and 6-keto-PGF1 alpha concentration in injury site (T13-L1) and adjacent cords (upper: T12, under: L2) were studied using a rat SCI model induced by Allen's weight drop method (50g-cm). The result showed that after SCI the SCBF in injury site significantly reduced during 1-2 hrs and reduced further during 4-8 hrs. The SCBF in adjacent cords also decreased during 4-8 hrs. TXB2 levels significantly increased at 1 hr and reached peak value at 4 hrs. The 6-keto-PGF1 alpha concentration also significantly increased at 1 hr and maintained that level for 24 hrs. The TXB2/6-keto-PGF1 alpha ratio was significantly elevated at 1 hr and reached its peak at 4 hrs after SCI, then gradually decreased to the preinjury level during 8-24 hrs. The negative correlation of SCBF with TXB2 concentration and TXB2/6-keto-PGF1 alpha ratio were appeared. The experimental results indicated that the imbalance of TXB2/6-keto-PGF1 alpha could be the main cause of microcirculatory disturbance and secondary damage in SCI.     

Muscarinic regulation of intracellular signaling and neurosecretion in gonadotropin-releasing hormone neurons

Agonist activation of cholinergic receptors expressed in perifused hypothalamic and immortalized GnRH-producing (GT1-7) cells induced prominent peaks in GnRH release, each followed by a rapid decrease, a transient plateau, and a decline to below basal levels. The complex profile of GnRH release suggested that acetylcholine (ACh) acts through different cholinergic receptor subtypes to exert stimulatory and inhibitory effects on GnRH release. Whereas activation of nicotinic receptors caused a transient increase in GnRH release, activation of muscarinic receptors inhibited basal GnRH release. Nanomolar concentrations of ACh caused dose-dependent inhibition of cAMP production that was prevented by pertussis toxin (PTX), consistent with the activation of a plasma-membrane Gi protein. Micromolar concentrations of ACh also caused an increase in phosphoinositide hydrolysis that was inhibited by the M1 receptor antagonist, pirenzepine. In ACh-treated cells, immunoblot analysis revealed that membrane-associated G(alpha q/11) immunoreactivity was decreased after 5 min but was restored at later times. In contrast, immunoreactive G(alpha i3) was decreased for up to 120 min after ACh treatment. The agonist-induced changes in G protein alpha-subunits liberated during activation of muscarinic receptors were correlated with regulation of their respective transduction pathways. These results indicate that ACh modulates GnRH release from hypothalamic neurons through both M1 and M2 muscarinic receptors. These receptor subtypes are coupled to Gq and Gi proteins that respectively influence the activities of PLC and adenylyl cyclase/ion channels, with consequent effects on neurosecretion.     

Nitric oxide as a regulator of prostacyclin synthesis in cultured rat heart endothelial cells

The effects of nitric oxide (NO) and its second messenger cyclic guanosine monophosphate (cGMT) on prostacyclin (PGI2) synthesis were studied in cultured rat heart endothelial cells using three different non-enzymatic nitric oxide releasing substances as well as inhibitors of nitric oxide synthase and of soluble guanylate cyclase. Production of prostacyclin, measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), was stimulated up to 1.7 fold in endothelial cells treated with the NO donors SIN-1 (3-morpholino sydnonimine), GEA 3162 (3-aryl-substituted oxatriazole imine) and GEA 3175 (3-aryl-substituted oxatriazole sulfonyl), chloride). In each case the synthesis of cGMP increase as much as 40-100 fold. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), decreased the basal production of 6-keto-PGF1 alpha in non-stimulated endothelial cells, an effect that could be reversed by the NO donors SIN-1, GEA 3162 and GEA 3175. cGMP formation in the L-NAME treated endothelial cells was unaltered. The guanylate cyclase inhibitors, methylene blue (100 mumol/l) and LY83583 (100 mumol/l), caused a 1.5-10 fold increase in 6-keto-PGF1 alpha production while NO-donor-stimulated endothelial cGMP production was decreased by 10 to 90%. However, when SIN-1 was used as a stimulant, LY83583 had no significant effect on the production of cGMP. These findings support the hypothesis that NO stimulates prostacyclin production directly by activating cyclooxygenase. The results also suggest that NO could have an indirect effect on prostacyclin production via cGMP.     

Prostaglandin I2 contributes to the vasodepressor effect of baicalein in hypertensive rats

Lipoxygenase inhibitors reduce blood pressure in hypertensive rats. The vasodepressor effect of lipoxygenase inhibitors may be related to increased production of prostaglandin (PG) I2 since lipoxygenase-derived fatty acid hydroperoxides inhibit PGI2 synthase. This hypothesis was examined in rats made hypertensive by infusion of angiotensin II (200 ng/min i.p.) for 12 to 14 days. In hypertensive but not in normotensive rats, the lipoxygenase inhibitor baicalein (60 mg/kg s.c.) increased (P<.05) the conversion of exogenous PGH2 to PGI2 by aortic segments, the release of 6-keto-PGF1alpha by aortic rings, the concentration of 6-keto-PGF1alpha in blood, and the renal excretion of 6-keto-PGF1alpha. Treatment with baicalein did not affect the blood pressure of normotensive rats but decreased the blood pressure of hypertensive rats from 177+/-8 to 133+/-9 mm Hg after 120 minutes (P<.05). Also, the lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (8 mg/kg s.c.) was without effect on the blood pressure of normotensive rats but decreased the blood pressure of hypertensive rats from 182+/-4 to 139+/-8 mm Hg (P<.05). However, the blood pressure of hypertensive rats pretreated with indomethacin (5 mg/kg i.v.) was affected by neither baicalein nor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. Moreover, in hypertensive rats in which baicalein had decreased blood pressure to 148+/-6 mm Hg, the administration of rabbit serum containing antibodies against 5,6-dihydro-PGI2 (0.3 mL i.v.) partially reversed the response to baicalein, increasing blood pressure to 179+/-7 mm Hg within 20 minutes (P<.05). The antibodies also were shown to block the vasodepressor effect of PGI2 but not of PGE2. Collectively, these data suggest contribution of PGI2 to the acute antihypertensive effect of baicalein in rats with angiotensin II-induced hypertension.     

Nuclear matrix proteins and their potential applications to diagnostic pathology

The sympathetic nervous system controls lipolysis in fat by activation of four adrenergic receptors: beta1, beta2, beta3, and alpha2. During pregnancy, maternal metabolism presents anabolic and catabolic phases, characterized by modifications of fat responsiveness to catecholamines. The contributions of the four adrenergic receptors to adipocyte responsiveness during pregnancy have never been studied. Our aim was to evaluate the influence of pregnancy on adrenergic receptor-mediated lipolysis in rabbit white adipocytes. Functional studies were performed using subtype-selective and non-selective adrenergic receptor agonists. Overall adrenergic responsiveness was measured with the physiological agonist epinephrine. Non-adrenergic agents were used to evaluate different steps of the lipolytic cascade. The alpha2- and beta1/beta2-adrenergic receptor numbers were determined with selective radioligands. Non-adrenergic agents revealed that pregnancy induced an intracytoplasmic modification of the lipolytic cascade in inguinal but not in retroperitoneal adipocytes. Pregnancy induced an increase in beta1- and specially beta3-mediated lipolysis. The amounts of adipocyte beta1/beta2- and alpha2-adrenergic receptors were increased in pregnant rabbits. Epinephrine effects revealed an increased contribution of alpha2-adrenergic receptor-mediated antilipolysis in adipocytes from pregnant rabbits. These results indicate that pregnancy regulates adipocyte responsiveness to catecholamines mainly via the alpha2- and beta3-adrenergic pathways. Pregnancy induces an intracytoplasmic modification of the lipolytic cascade, probably via hormone-sensitive lipase, with differences according to fat location.-Bousquet-Mélou, A., C. Mu?oz, J. Galitzky, M. Berlan, and M. Lafontan. Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells.     

Influence of the estrous cycle on the norepinephrine-induced contraction of rat aorta: relationship to vascular prostanoids biosynthesis

Since ovarian sex steroids (estradiol and progesterone) may affect both blood pressure and prostanoids synthesis, and because prostaglandin-E2 (PGE2) and prostacyclin (PGI2) can modulate the vascular action of pressor hormones, we investigated the vascular reactivity to norepinephrine during the estrous cycle of the rat. In addition, we determined the vascular biosynthesis of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2) at different stages of the estrous cycle. Cumulative dose-response curves were obtained by a stepwise increase in the concentration of norepinephrine. The contraction of thoracic aortic rings induced by norepinephrine did not change significantly between estrus, metestrus and diestrus. However, aortic rings obtained on proestrus showed a significant reduction in the maximal contraction (Emax) induced by norepinephrine (p < 0.001). In addition, we found significant increases in vascular synthesis of PGE2 and PGI2 on proestrus (p < 0.001). These results indicate that vascular reactivity and vascular prostanoids synthesis are influenced by the hormonal changes occurring during the estrous cycle of normal female rats. It is possible that prostanoids generated locally may play an important role in the regulation of vasomotor tone in the systemic vascular bed throughout the estrous cycle.