Regional differences in the responses to prostanoids of circular muscle from guinea-pig isolated intestine

The effects of prostaglandins (PGs) D₂, E₂, F_2α, an epoxymethano analogue of PGH₂ (U-46619), prostacyclin (PGI₂), 6-keto-PGF_lα and thromboxane (Tx) B₂ were tested on spirally-cut strips of guinea-pig isolated ileum or colon. In the ileum no prostanoid exerted a marked effect on the resting tissue, but PGD₂, PGE₂ or PGI₂ 1 μg ml^?1 inhibited submaximal contraction to KC1. U-46619 1 μg ml^?1 either inhibited or increased contractions to KC1, but PGF_2α, 6-keto-PGF_1α or TxB₂ 1 μg ml^?1 had no significant effect. PGE₂ relaxed colonic strips whereas the other prostanoids caused contraction, except for TxB₂ which had no effect. The PG antagonist SC-19220 blocked colonic contractions to the prostanoids, and a residual inhibitory effect of PGD₂, U-46619 or PGI₂ was demonstrated by the reduction of submaximal contractions to acetylcholine. Our results suggest that prostanoid receptors mediating inhibitory responses of circular muscle predominate in the ileum, whereas in the colon both excitatory and inhibitory prostanoid receptors occur.     

Pericardial release of prostacyclin induced by bradykinin and angiotensin II: effects on coronary blood flow in the dog

Angiotensin II and bradykinin are potent releasers of prostanoids, and it has been suggested that these prostanoids may modulate vascular tone in a number of organs. We previously showed that the pericardium is an important site of prostacyclin biosynthesis, and in this study we have investigated whether or not prostacyclin released into the pericardial fluid influences coronary blood flow. Angiotensin II and bradykinin were infused intra-aortically into anaesthetized dogs, and coronary blood flow in the circumflex artery was measured with electromagnetic probes. Krebs solution irrigating the pericardial surfaces was monitored for prostanoid release using a cascade of bioassay tissues as previously described. Angiotensin II infusions (25 ng/kg/min) increased arterial blood pressure, decreased coronary blood flow, and increased the release of a prostacyclin-like substance into the pericardial irrigating fluid. Inclusion of indomethacin (1 microgram/ml) in the pericardial irrigating fluid abolished angiotensin II-induced release of the prostacyclin-like substance from the heart, did not affect resting coronary flow, but potentiated the coronary vasoconstrictor response to intra-arterial angiotensin II. Bradykinin infusions (0.2 microgram/kg/min) also released the prostacyclin-like substance into the pericardial fluid, and caused a transient decrease in arterial pressure and increase in coronary blood flow. Inclusion of captopril (1 microgram/ml) in the irrigating fluid increased slightly the release of the prostacyclin-like substance, but did not alter the increase in coronary blood flow produced by bradykinin. Moreover, when prostacyclin release was abolished by pericardial indomethacin, the coronary vasodilator response to bradykinin was not altered. A large intravenous dose of indomethacin (5 mg/kg) increased the coronary vasoconstrictor response to angiotensin II, but, again, did not alter the vasodilator response to bradykinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

EFFECTS OF β-ADRENOCEPTOR BLOCKADE ON PROSTACYCLIN MEDIATED RENIN RELEASE IN SHEEP

1. The effect of prostacyclin (PGI₂) infusion on plasma renin concentration (PRC) was examined before and after propranolol treatment in sheep. 2. Increasing doses of prostacyclin (0.05, 0.1 or 0.3 μg/kg per min) produced dose dependent increases in PRC. 3. There was a significantly lower PRC response after propranolol at 0.3 μg/kg per min only.     

Use of the rabbit transverse stomach-strip to identify and assay prostacyclin,PGA2, PGD2 and other prostaglandins

The preparation and use of the rabbit transverse stomachstrip as an isolated assay tissue for prostaglandins is described. The tissue is relaxed by low concentrations of PGE₂, PGA₂ and PGD₂ and contracted by prostacyclin (PGI₂) and PGF_2α. The decomposition product of prostacyclin, 6-oxo-PGF_1α, has little effect, whereas the endoperoxde PGH₂ and thromboxane A₂ cause a small contraction. Arachidonic acid also contracts the tissue and this response is abolished by indomethacin. This tissue also responds to low concentrations of histamine, acetylcholine, noradrenaline and bradykinin. In conjunction with other bioassay tissues such as the rat stomach strip and rabbit coeliac artery, the rabbit transverse stomach strip provides a convenient method of detection or identification of known arachidonic acid metabolites.     

EFFECT OF INDOMETHACIN ON RESPONSES OF SHEEP ISOLATED CORONARY ARTERY TO ARACHIDONIC ACID

Indomethacin (2.8 μmol/1) did not consistently affect basal tone of sheep coronary artery strips, while a ten-fold higher concentration increased tension in 50% of the preparations tested. When acetylcholine was used as a spasmogen, oscillations in induced tone and relaxations produced by arachidonic acid (6.6 μmol/1) were abolished by indomethacin, 2.8 μmol/1 and 7 μmol/1, respectively. Prostacyclin (PGI₂) and prostaglandin E₁ decreased and PGE₂ increased arterial tension while PGF_2α was inactive. Responses to PGI₂ were reduced by indomethacin (28 μmol/1) but not by indomethacin (2.8 μmol/1). It is suggested that sheep isolated coronary arteries synthesize and release prostacyclin in the presence of acetylcholine and arachidonic acid and that such synthesis can be inhibited by indomethacin.     

UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE₂, PGF_2α, PGD₂ and PGI₂ (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE₂, PGD₂ and the PGD₂ metabolite PGJ₂. Twenty-four hours after treatment with UVB (25 mJ/cm²), production of PGE₂ and PGJ₂ increased, while PGD₂ production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5–25 mJ/cm²) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE₂ (EP1 and EP2), PGD₂ (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.     

CAPTOPRIL POTENTIATES DEPRESSOR RESPONSES TO ARACHIDONIC ACID IN THE RAT

1. In chloralose anaesthetized rats intravenous administration of captopril (0.5 mg/kg) was followed by an approximately 100-fold decrease in sensitivity to the pressor actions of angiotensin I. Concomitantly there was a 100-fold increase in sensitivity to the depressor effects of bradykinin. 2. Depressor responses to intravenous prostacyclin (PGI₂), prostaglandin E₂ (PGE₂) or a low dose of arachidonic acid (1 mg/kg) were not changed by captopril administration, but responses to a high dose of arachidonic acid (3 mg/kg), given either intravenously or into the aortic arch, were enhanced for up to two hours afterwards. 3. Depressor responses to arachidonic acid, both before and after captopril, were inhibited after intravenous indomethacin (1 mg/kg). 4. These results support the hypothesis that increased synthesis of prostaglandins in the circulation contributes to the hypotensive action of captopril.     

Prostaglandin production by intact isolated gastric parietal cells

An enriched population of isolated gastric parietal cells was obtained from canine gastric mucosa. Parietal cells incubated with [¹⁴C]arachidonic acid produced radiolabelled PGF_2α. PGE₂, PGD₂ and 6-keto PGF_1α (acid hydrolysis product of PGI₂). The prostaglandin synthesis was inhibited by indomethacin. Prostaglandin production, as measure by gas chromatography-mass spectrometry demonstrated that PGF_2α was produced in the highest quantity followed by PGE₂ and 6-keto PGAF_1α. This demonstrates that isolated parietal cells are capable of producing prostaglandins. Since prostaglandins have a potent effect on gastric acid secretion, local prostaglandin synthesis could modulate parietal cell function.     

EFFECTS OF PROSTAGLANDIN(PG) E2, D2 I2, 6-KETO-PGF1α AND ARACHIDONIC ACID ON EXCITATORY TRANSMISSION OF CANINE SMALL INTESTINE

• 1 Effects of prostaglandin(PG) E₂, D₂, I₂, 6-keto-PGF_1α and arachidonic acid on the excitatory transmission were examined in the separated longitudinal and circular muscles of canine small intestine in vitro.
• 2 Both muscles contracted in response to electrical field stimulation (1 msec, 20 Hz, 100 pulses, supramaximal voltage: indirect stimulation). The contractions were abolished by treatment with tetrodotoxin (0.3 μM). In the presence of tetrodotoxin, the muscle contracted in response to stimulation using a pulse duration of 20msec (direct stimulation). Sites of action of drugs were determined by the difference in their effects on the responses to indirect and direct stimulation.
• 3 PGE₂ strongly inhibited the contractile response of circular muscle to indirect stimulation whilst slightly inhibiting that to direct stimulation. The data suggest that PGE₂ may act on the transmission prejunctionally and reduce the transmitter release in circular muscle.
• 4 PGD₂ and PGI₂ in high concentrations slightly inhibited the contractile responses of circular muscle to both indirect and direct stimulations, indicating that PGD₂ and PGI₂ slightly inhibited the transmission postjunctionally. 6-keto-PGF_1α did not inhibit the transmission of circular muscle.
• 5 Arachidonic acid inhibited the contractile response of circular muscle to indirect stimulation, and indomethacin attenuated the arachidonic acid-induced inhibition. PGE₂, biosynthetized from arachidonic acid, may act on the transmission of circular muscle.
• 6 In the longitudinal muscle, none of PGE₂, PGD₂, PGI₂ or 6-keto-PGF_1α inhibited the contractile response to indirect stimulation. 7 Arachidonic acid slightly inhibited the contractile response of longitudinal muscle to indirect stimulation. However, indomethacin did not antagonize this effect. 8 The results suggest that the transmission of circular muscle in canine small intestine is prejunctionally regulated by PGE₂, and that of longitudinal muscle is slightly affected by arachidonic acid or by metabolites through lipoxygenase.

     

苯妥英钠与前列腺素E2联合应用抗实验性哇巴因心律失常的研究

关于联合用药治疗心律失常,目前报道甚少,对此问题深入地进行基础理论或临床治疗学的研究,具有实际意义。已知苯妥英钠(DPH)为治疗洋地黄心律失常的有效药物。近年来亦发现前列腺素E(PGE)有抗哇巴因心律失常的作用,但其机制待深入探讨。一般认为洋地黄心律失常与交感神经活动增强有关,并发现洋地黄致心律失常时心肌儿茶酚胺含量     

Stimulation of prostacyclin release from the epicardium of anaesthetized dogs.

1 The generation of prostanoids in the hearts of anaesthetized dogs was studied by irrigating in situ the epicardial surface with Krebs solution. Prostanoids were measured by direct bioassay on smooth muscles and by radioimmunoassay of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) and prostaglandin E2 (PGE2) in the epicardial irrigation fluid. 2 The epicardial irrigation fluid contained a prostacyclin-like substance, as indicated by the bioassay tissues, and immunoreactive 6-oxo-PGF1 alpha; PGE2-like materials were also detected. By both methods the output of the prostacyclin-like substance, which decreased with time of epicardial irrigation, was increased by manipulating the heart and by adding arachidonic acid (3 microgram/ml), and decreased by adding indomethacin (1 microgram/ml) to the irrigation fluid. 3 Bioassayed prostacyclin and immunoreactive 6-oxo-PGF1 alpha in the epicardial irrigation fluid increased by about 3-5 ng/ml during and after infusion of isoprenaline (0.1 microgram kg-1 min-1). The substance was not released by isoprenaline when indomethacin was added to the irrigation fluid, or when propranolol (0.5 mg/kg) was given intravenously. 4 Aortic constriction, bilateral carotid artery occlusion and intravenous angiotensin infusion all increased output of the prostacyclin-like substance into the epicardial irrigation fluid. The output was abolished by treating the heart with indomethacin (10 mg/kg intravenously or 1 microgram/ml epicardially). 5 The prostacyclin-like substance was also released by all of the above stimuli after the parietal pericardium had been removed and replaced by a plastic sheet. 6 It is concluded that prostacyclin is continually released from tissues close to the epicardial surface and from the pericardium, and that prostacyclin generation increases when cardiac workload increases. Prostacyclin of epicardial or pericardial origin might therefore contribute to metabolic regulation of coronary blood flow.     

Prostaglandin inhibition of amphetamine-induced circling in mice

The effect of prostaglandins (PG) on amphetamine(AMPH)-induced circling was examined in mice unilaterally lesioned with 6-hydroxy-dopamine. At doses of 0.03–1.0 nmol/g, intraventricularly injected PGD₂, PGE₂, and PGF₂ all inhibited AMP-induced circling, while thromboxane-B₂ (TxB₂) was inactive at 1.0 nmol/g. The inhibition of circling was not due to alterations in body temperature as measured by rectal temperature changes. When injected intrastriatally, the same major PG inhibited AMP-induced circling at the lower doses of 0.01–0.1 nmol/g, while the PGE₂ metabolite 13,14-dihydro-15-keto-PGE₂ was inactive at 0.1 nmol/g. PG administered alone did not procude circling. For both routes of administration, the order of potency was PGE₂>PGD₂>PGF₂. These results suggest that PG can alter motor function governed by central dopaminergic pathways.     

The Protective Effects of Up-Regulating Prostacyclin Biosynthesis on Neuron Survival in Hippocampus

Cellular arachidonic acid (AA), an unsaturated fatty acid found ubiquitously in plasma membranes, is metabolized to different prostanoids, such as prostacyclin (PGI₂) and prostaglandin E₂ (PGE₂), by the three-step reactions coupling the upstream cyclooxygenase (COX) isoforms (COX-1 and COX-2) with the corresponding individual downstream synthases. While the vascular actions of these prostanoids are well-characterized, their specific roles in the hippocampus, a major brain area for memory, are poorly understood. The major obstacle for its understanding in the brain was to mimic the biosynthesis of each prostanoid. To solve the problem, we utilized Single-Chain Hybrid Enzyme Complexes (SCHECs), which could successfully control cellular AA metabolites to the desired PGI₂ or PGE₂. Our in vitro studies suggested that neurons with higher PGI₂ content and lower PGE₂ content exhibited survival protection and resistance to Amyloid-β-induced neurotoxicity. Further extending to an in vivo model, the hybrid of PGI₂-producing transgenic mice and Alzheimer’s disease (AD) mice showed restored long-term memory. These findings suggested that the vascular prostanoids, PGI₂ and PGE₂, exerted significant regulatory influences on neuronal protection (by PGI₂), or damage (by PGE₂) in the hippocampus, and raised a concern that the wide uses of aspirin in cardiovascular diseases may exert negative impacts on neurodegenerative protection.

Our study intended to understand the crosstalk of prostanoids in the hippocampus, a major brain area impacted in AD, by using hybrid enzymes to redirect the synthesis of prostanoids to PGE₂ and PGI₂, respectively. Our data indicated that during inflammation, the vascular mediators, PGI₂ and PGE₂, exerted significant regulatory influences on neuronal protection (by PGI₂), or damage (by PGE₂) in the hippocampus. These findings also raised a concern that the widely uses of non-steroidal anti-inflammatory drugs in cardiovascular diseases may exert negative impacts on neurodegenerative protection.

     

INCREASED CONVERSION OF ARACHIDONIC ACID TO VASODILATOR PROSTANOIDS IN SPONTANEOUSLY HYPERTENSIVE RATS

1. Vasodepressor responses to intravenous injection of prostacyclin, arachidonic acid, and nitroprusside were examined in anaesthetized, spontaneously hypertensive rats (SHR) of the Okamoto strain, and in their normotensive Wistar– Kyoto (WKY) controls. 2. Depressor responses to prostacyclin and nitroprusside did not differ significantly between the two strains. 3. The vasodepressor effects of arachidonic acid were greater and much more prolonged in SHR than in WKY. In rats treated with indomethacin (2 mg/kg) arachidonic acid induced only transient depressor responses which did not differ significantly between these strains. 4. It is concluded that SHR do not differ from WKY in their sensitivity to prostacyclin but they have enhanced ability to transform exogenous arachidonic acid into vasodilator prostanoids.     

Latest discoveries in prostaglandin receptor modulators

Prostanoids (prostaglandins and the thromboxanes) are cyclooxygenase products derived from C-20 unsaturated fatty acids. Since arachidonic acid is the most abundant among these precursor fatty acids in mammals, including humans, the series 2 prostanoids are predominantly formed in the body. Thus, cyclooxygenases metabolise arachidonate to five primary prostanoids: PGE₂, PGF_{2 α}, PGI₂, TXA₂ and PGD₂. These lipid mediators interact with specific members of a family of distinct G-protein-coupled prostanoid receptors, designated EP, FP, IP, TP and DP, respectively. As a review focused on latest discoveries and developments of novel TXA₂ modulators patented from January 1997 to December 2000 has been recently published, this paper specifically focuses on newly developed IP, DP, FP and EP modulators patented for three years, from January 1998 to December 2001. Indeed, because prostaglandins are involved in a large series of pathophysiological processes, a classification of these modulators has been proposed, based on the type of receptors activated or inhibited. Their pharmacological profile is also described.     

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [³H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg⁹-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE₂) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca²⁺ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE₂ formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE₂ formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.     

STIMULATION BY ANGIOTENSIN OF PROSTACYCLIN BIOSYNTHESIS IN RATS AND DOGS

1. Stimulation of prostanoid release by angiotensins (AI and All) in rat isolated mesenteric vasculature and in the circulation of anaesthetized dogs has been investigated by bioassay. 2. AI and All released a PGI₂-like substance into rat mesenteric effluent and arterial blood of dogs; PGE₂, PGF_2α or TXA₂ were not detected. 3. AI stimulated PGL release in both systems largely as a result of its conversion to All, since PGI₂ release was much reduced after treatment with captopril. 4. Intravenous AII (0-2-1.0μg kg^?1min^?1) in dogs released PGI₂ mainly from the lungs since right atrial blood contained much less than arterial blood. 5. Indomethacin (1 μg/ml) abolished All-induced PGI₂ release from the mesentery preparation, but intravenous indomethacin (10 mg/kg), meclofena-mate (2 mg/kg) or aspirin (100 mg/kg) did not eliminate the pulmonary source of PGI₂ in dogs. These findings highlight the dangers of assuming in vivo treatment with cyclo-oxygenase inhibitors abolishes biosynthesis of all prostanoids.     

Prostaglandin inhibition of apomorphine-induced circling in mice

The effect of prostaglandins (PGs) on apomorphine (apo)-induced circling was examined in unilaterally lesioned mice. Intraventricularly injected PGD₂, PGE₂, and PGF_2α at a dose of 1.0 nmole/g all inhibited apo-induced circling. When injected directly into the striatum, these same PGs also inhibited circling in a dose range of 0.01–0.1 nmole/g, while the PGE₂ metabolite, 13, 14-dihydro-15-keto-PGE₂, was inactive at 0.1 nmole/g. For both routes of administration, PGF_2α appeared to be the most potent of the PGs tested. PGs administered alone by either route to unilaterally lesioned mice did not produce circling. Pretreatment with the PG synthetase inhibitor, indomethacin, caused the apo treated mice to circle at significantly higher rates than control animals. These results are the first report suggesting that within dopamine (DA)-mediated pathways PGs act at sites postsynaptic to the dopaminergic synapse.     

Bradykinin induces NO and PGF2α production via B2 receptor activation from cultured porcine basilar arterial endothelial cells

Our previous in vitro study demonstrated that bradykinin (BK) induced relaxation and contraction of porcine basilar artery (PBA) mediated via activation of endothelial B₂ receptors. The main relaxing and contracting factors appeared to be nitric oxide (NO) and prostaglandin (PG) H₂, respectively, but not thromboxane A₂. After obtaining these findings, we succeeded in cultivating endothelial cells isolated from the PBA. Although PGH₂ has different functionally active isoforms, including PGD₂, PGE₂, and PGF_2α, we have not yet clarified which of them is responsible for BK-induced contraction. Therefore, we attempted to quantify NO and PG production from cultured porcine basilar arterial endothelial cells (PBAECs) and to identify which of the PGs was involved in this contraction. The cultured PBAECs produced NO spontaneously, and BK enhanced this production in a concentration-dependent manner. The NO synthase inhibitor Nω-nitro-l-arginine (L-NNA) and the B₂ receptor antagonist HOE-140, but not the B₁ receptor antagonist des-Arg⁹, [Leu⁸]-BK, completely abolished it. In a functional study, PGD₂, PGE₂, and PGF_2α induced concentration-dependent contractions in isolated porcine basilar arterial rings, the order of maximum contraction being PGF_2α?>?PGE₂?>?PGD₂. The cultured PBAECs produced PGD₂, PGE₂, and PGF_2α spontaneously, and BK significantly enhanced the production of PGF_2α, but not that of PGD₂ and PGE₂. The B₂, but not B₁, antagonist completely abolished the BK-enhanced production of PGF_2α. These results suggest that BK induces production of NO and PGF_2α simultaneously from PBAECs via B₂ receptor activation.     

非普拉宗对环氧酶活性的影响

目的　研究非普拉宗(feprazone, Fep)对环氧酶-1和环氧酶-2活性的影响。 方法　用放免法测定PGE2含量反映环氧酶-2活性,测定6-酮-前列腺素F_1α含量反映环氧酶-1活性。 结果　Fep在0.1,1.0,10.0 μmol.L^-1能剂量依赖性抑制小鼠腹腔巨噬细胞PGE₂生成,在相同浓度下对小牛主动脉内皮细胞6-keto-PGF_1α生成抑制作用较弱。结论　Fep显著抑制PGE₂生成,对PGI₂生成影响较小,提示其对环氧酶-2有较强的抑制作用。