Failure of anti-inflammatory steroids to inhibit prostaglandin production by rat polymorphonuclear leucocytes.

1 Like rabbit polymorphonuclear (PMN) leucocytes, rat peritoneal glycogen-induced PMN leucocytes produced much greater amounts of prostaglandin when incubated with killed bacteria than in the absence of phagocytosable material. 2 Rat PMN leucocytes mainly prostaglandin E2 (PGE2), in amounts up to 17 ng/10(6) cells in 90 min incubation, some 25 times the amount produced by resting cells. 3 Indomethacin and meclofenamic acid inhibited prostaglandin production by resting and phagocytosing cells, the IC50 being of the order of 10(-6) to 10(-7) M for both drugs. 4 Hydrocortisone and dexamethasone at concentrations up to 10(-4) M did not cause significant dose-related inhibition of prostaglandin production in this system. 5 It is suggested that the phagocytosing PMN leucocyte is insensitive to the action of anti-inflammatory steroids with respect to prostaglandin production.     

Possible influence of intrarenal generation of kinins on prostaglandin release from the rabbit perfused kidney.

The effects of bradykinin and kininogen on renal prostaglandin release were studied in rabbit isolated kidneys perfused with oxygenated Krebs solution. The concentration of prostaglandin-like material in kidney effluent was determined by bioassay after extraction of the samples with organic solvents. In 7 experiments the samples were assayed after separation of prostaglandins E and F by thin layer chromatography. Addition of bradykinin to the perfusing fluid increased the venous and urinary effluxes of prostaglandin E-like substance by sixfold and fivefold, respectively, but efflux of prostaglandin F-like material was unaffected. Addition of kininogen to the perfusing fluid augmented the venous and urinary release of prostaglandin E-like substances by fifteenfold and ninefold respectively and caused a twofold increase in the efflux of prostaglandin F-like material into the venous effluent. Aprotinin, a kallikrein inhibitor, reduced the prostaglandin releasing action of kininogen but not of bradykinin. In contrast, inhibition of prostaglandin synthesis by indomethacin suppressed the release of prostaglandin evoked by either bradykinin or kininogen. This study suggests that augmented release of prostaglandins in response to kininogen is a consequence of renal generation of kinins. Thus, changes in the intrarenal activity of the kallikreinkinin system may modulate renal prostaglandin release.     

In vivo inhibition of prostaglandin synthesis in rabbit kidney by non-steroidal anti-inflammatory drugs

To define dose- and time-response properties for in vivo inhibition of renal prostaglandin (PG) synthesis, aspirin, diclofenac sodium, indomethacin and d-naproxen were injected intravenously in different doses to unanaesthetized rabbits. After 30 min. the animals were killed and the post mortem accumulation of PGE2 and PGF2alpha in the renal medulla was determined by mass fragmentography. In control animals, the accumulated levels of PGE2 and PGF2alpha in the medulla were 9.2+/-2.2 (S.D.) and 1.5+/-0.6 microgram/g, respectively. Dose-dependent inhibition was demonstrated with all the drugs. The ED95 was for aspirin 15 mg/kg, for diclofenac sodium 1.5 mg/kg, for indomethacin 1.5 mg/kg and for d-naproxen 5 mg/kg. The duration of inhibition was studied by radioimmunoassay in anaesthetized rabbits by following the urinary excretion of PGF2alpha and PGE2 after an intravenous injection of solvent or test drug in doses twice the ED95. For three hours following aspirin, diclofenac sodium, indomethacin and d-naproxen, the decreases in urinary excretion of PGF2alpha ranged from 64 to 88, 87 to 95, 64 to 90 and from 40 to 77 per cent of the control, respectively, and the decreases in PGE2 excretion were of similar magnitude. Together these results indicate that diclofenac sodium might be used as a long-lasting and potent alternative to indomethacin and aspirin in experimental studies on the renal PG system in vivo.     

Antipyretic analgesics inhibit prostaglandin release from astrocytes and macrophages similarly

The effect of acid and non-acid antipyretic analgesics on prostaglandin (PG) release from cultured mouse astrocytes and peritoneal macrophages was investigated in order to test the hypothesis that the non-acid compounds are more potent inhibitors of PG formation in brain than in peripheral tissues. Stimulation of the cells by the divalent cation ionophore A 23187 (10(-6) mol/l) induced PG release from astrocytes and macrophages (mainly PGD2 and PGE2, respectively). This PG release was inhibited by acetylsalicylic acid (10(-5) - 10(-6) mol/l) and indomethacin (10(-6) - 10(-9) mol/l) but also by high concentrations (10(-3) - 10(-5) mol/l) of the non-acid compounds 4-methyl-aminophenazone, the main active metabolite of dipyrone (metamizol), and acetaminophen (paracetamol). No difference was found in the inhibitory potency of the drugs in astrocytes and macrophages, suggesting that a specific sensitivity of brain cells toward non-acid antipyretic analgesics does not contribute to their analgesic effect.     

Inhibition of transmitter release from isolated rabbit kidney by bradykinin.

The effect of kallikrein on the release of norepinephrine has been studied in the isolated perfused rabbit kidney. Kallikrein in the perfusion medium caused an inhibition in the rise of the perfusion pressure induced by periarterial stimulation and in the contraction of rabbit aorta superfused by the venous outflow of the stimulated kidney. On the contrary, kallikrein caused a potentiation in the urine volume induced by periarterial stimulation. Similar findings were obtained when bradykinin was added instead of kallikrein. Kallikrein did not alter the pressure response and rabbit aorta contraction but potentiated the diuretic effects of exogenously administered norepinephrine. Acetyl salicyclic acid when added to the perfusion medium containing kallikrein caused a reversal in all parameters measured in this study. It is concluded that the changes of the effects of periarterial stimulation of the isolated rabbit kidney by kallikrein are mediated through the bradykinin-mediated increased generation of intrarenal prostaglandins.     

Effect of tiopronin on prostaglandin synthesis in rabbit kidney medulla slices

The effect of 2-mercaptopropionylglycine (tiopronin), which is widely used for the treatment of various hepatic disorders, on the generation of medullary prostaglandins (PG) E2 and F2 alpha has been examined. Tiopronin had a potent inhibitory effect on PG E2 formation. Simultaneously, PG F2 alpha production was increased. In the presence of tiopronin the net increased amount of PG F2 alpha was much smaller than the net decreased amount of PG E2 (6-20%). These results suggest that tiopronin has the potential to modulate PG E2 and F2 alpha synthesis by affecting endoperoxide E2 isomerase or endoperoxide reductase and that this effect may represent some pharmacological action of the drug.     

Inhibition of prostaglandin synthesis in rabbit kidney medulla slices by antioxidants

The inhibitory effects of NN'-diphenyl-p-phenylenediamine (DPPD), sodium diethyldithiocarbamate (SDDC) and 2,6-di-t-butylphenol (DTBP) on the generation of medullary prostaglandin E have been compared. DPPD (1 μm) and SDDC (1 mm) failed to inhibit arachidonic acid-induced stimulation of prostaglandin E synthesis, while DTBP (1 mm) inhibited it. This suggests that DPPD and SDDC inhibit prostaglandin formation by affecting a phospholipase pathway, and the inhibition of prostaglandin generation by DTBP occurs at the cyclooxygenase step.     

Effect of pyridoxine on prostaglandin synthesis in rabbit kidney medulla slices

Pyridoxine stimulated the generation of prostaglandin E₂ in rabbit kidney medulla slices. Moreover, pyridoxine in the presence of aspirin reduced the release of linoleic acid, but enhanced the release of arachidonic acid from the medulla slices as compared with aspirin alone, indicating that the enhancement of prostaglandin E₂ formation elicited by pyridoxine may be ascribed to an increased conversion of linoleic acid to arachidonic acid. These results suggest that pyridoxine can be an important modulating factor in prostaglandin synthesis by the kidney.     

Renin release from the ischaemic kidney

1. We have confirmed that spinal section, and also pithing, inhibits the pressor response associated with the release of renin that follows re-establishment of circulation to the ischaemic rat kidney. Brain transections at bulbar and midthalamic levels did not modify the blood pressure elevation.2. Several pharmacological antagonists of the sympathetic nervous system did not modify the blood pressure response. These observations are not consistent with the view that a neural element is necessary for renin release.3. Constant flow perfusion of the ischaemic kidney in the intact and spinal sectioned rat was performed to evaluate haemodynamic factors involved in renin release. The pressor response was present in spinal sectioned animals under these conditions.4. These results suggest that the nervous system is necessary to maintain adequate blood flow for renin ;washout' into the systemic circulation rather than to release renin from juxtaglomerular cells.     

The release of prostaglandins and rabbit aorta contracting substance (RCS) from rabbit spleen and its antagonism by anti-inflammatory drugs

1. Slices of rabbit spleen generate rabbit aorta contracting substance (RCS) and a prostaglandin of the E series when they are vibrated or stirred.

2. The release of both substances is increased by arachidonic acid and inhibited by aspirin-like drugs.

3. When the spleen effluent containing RCS and prostaglandin E is incubated for a further 3 min, the prostaglandin concentration increases and the RCS concentration declines.

4. RCS may therefore be an unstable intermediate in the biosynthesis of prostaglandins.

     

Pharmacological manipulation of cyclo-oxygenase-2 in the inflamed hydronephrotic kidney.

1. Bradykinin (BK, 1 microgram) caused a small (2 fold at 6 h) increase in prostaglandin E2 (PGE2) in the normal rabbit kidney, perfused ex vivo. This was exaggerated (6 fold at 6 h) in the hydronephrotic kidney (HNK). The exaggerated release of PGE2 was attenuated by cycloheximide, an inhibitor of protein synthesis or by dexamethasone, a steroid known to inhibit the induction of cyclo-oxygenase (COX-2). BK (1 microgram) when injected at 6 h of perfusion increased the release of PGE2 from 90 +/- 33 pg ml-1 min-1 to 3069 +/- 946 pg ml-1 min-1. This was reduced to 200 +/- 30 pg ml-1 min-1 in kidneys infused with cycloheximide (1 microM) and to 250 +/- 40 pg ml-1 min-1 in kidneys infused with dexamethasone (n = 8). 2. When tested on human and murine recombinant COX-1 and COX-2 enzymes, DuP-697 was at least 50 fold more selective for COX-2 than for COX-1. 3. DuP-697 reduced the exaggerated release of PGE2 elicited by BK in the HNK (e.g., at 6 h of perfusion BK-evoked PGE2 release decreased from 3069 +/- 946 pg ml-1 min-1 to 187 +/- 22 pg ml-1 min-1 after perfusion with 1 microM DUP-697, n = 8). 4. Cycloheximide, dexamethasone or DuP-697 at doses used to inhibit completely the exaggerated release of PGE2 in the hydronephrotic kidney, failed to inhibit the release of PGE2 elicited by the injection of BK (1 microgram) in the normal contralateral kidney. 5. Indomethacin (1 microM), a non-selective COX-1 and COX-2 inhibitor, completely inhibited PGE2 release in the normal contralateral as well as in the hydronephrotic kidney. 6. We suggest that renal prostaglandin production in the normal kidney is driven by the activity of constitutive COX-1 while at sites of inflammation, such as the hydronephrotic kidney, there is induction of COX-2 that can be blocked selectively by anti-inflammatory glucocorticoids or selective COX-2 inhibitors.     

Purinergic vs peptidergic stimulation of lipolysis and prostaglandin generation in the perfused rabbit kidney

Intact perfused rabbit kidney contains three different systems for generation of arachidonate oxygenated products. Each of these systems is associated with its specific type of activating agonist; the agonists are the vasoactive peptide hormones bradykinin and angiotensin II, the adenine nucleotides ATP and ADP, and exogenous arachidonic acid. The three systems are clearly distinguished by several biochemical parameters which include the type of lipolysis induced and the extent of coupling between the hydrolyzed arachidonic and its conversion to specific oxygenated products. The highest degree of selectivity in the lipolytic process and in the coupling to PGE₂ generation is seen following stimulation with bradykinin or angiotensin II. These hormones induce the hydrolysis of only arachidonic and PGE₂ is the major product. Furthermore, arachidonate hydrolysis is from a unique lipid pool which is characterized by a slow turnover of arachidonic. The entire process of lipolysis and prostaglandin E₂ synthesis is terminated within 1 min after stimulation and is followed by a hormone-induced re-acylation process in which excess released arachidonate is re-esterified into cellular lipids. The adenine nucleotides ATP and ADP induce a less selective lipolytic reaction which results in the hydrolysis of arachidonic and linoleic acids. This lipolytic process is less coupled to arachidonic oxygenation as evident from the 1 min delay between arachidonate release and prostaglandin generation. Arachidonate released by the nucleotides orginates from a lipid pool which has a higher turnover and readily incorporates exogenous acid. Generation of oxygenated products from administered exogenous acid is the least coupled process with apparent conversion of only 1–4% to prostaglandin products, amongst which 6-keto-PGF_1α predominates.     

Inhibitors of phosphodiesterase 5 (PDE 5) inhibit the nerve-induced release of nitric oxide from the rabbit corpus cavernosum

BACKGROUND AND PURPOSE: Nitrergic neurons are important for erectile responses in the corpus cavernosum and impaired signalling results in erectile dysfunction, today treated successfully by oral administration of the selective phosphodiesterase 5 (PDE 5) inhibitors sildenafil, tadalafil and vardenafil. Although the importance of nitrergic neurons in urogenital function has become evident, it has not been investigated if the PDE 5 inhibitors affect the nerve-induced release of nitric oxide (NO). In a previous study we found that the soluble guanylate cyclase (sGC)/cyclic guanosine 3',5'-monophosphate (cGMP) pathway might modulate nerve-induced release of NO in isolated cavernous tissue. EXPERIMENTAL APPROACH: Electrical field stimulation (EFS 5 Hz, 40 V, 0.3 ms pulse duration, 25 pulses at intervals of 2 min) of rabbit isolated cavernous tissue elicited reproducible, nerve-mediated relaxations in the presence of scopolamine (10(-5) M), guanethidine (10(-5) M) and phenylephrine (3 x 10(-6) M). In superfusion experiments, nerve stimulation (20 Hz, 40 V, 1 ms) of the cavernous tissue evoked release of NO/NO2-, measured by chemiluminescence. KEY RESULTS: Sildenafil, tadalafil and vardenafil decreased the muscular tone and prolonged the relaxations to nerve stimulation. The evoked release of NO decreased to 72+/-11%, 55+/-16% and 61+/-14% of control, respectively after addition of sildenafil, tadalafil or vardenafil (all 10(-4) M, n=6-8, p<0.05). CONCLUSIONS AND IMPLICATIONS: Selective PDE 5 inhibitors influence the nerve-induced release of NO, probably via cGMP-mediated negative feedback. This negative feedback might explain why priapism is not seen during monotherapy with the PDE inhibitors.     

Inhibition by non-steroidal anti-inflammatory agents of the release of rabbit aorta contracting substance and prostaglandins from chopped guinea-pig lungs

The release of prostaglandins (PGs) and rabbit aorta contracting substance (RCS) was investigated using mechanical agitation of chopped lung tissue from unsensitized guinea-pigs. Manual stirring of the lung tissue for 45 s produced maximal release of prostaglandins, a release corresponding to the effect of [20–40 ng ml^?1 of PGE₂ x 45 s at 5 ml min^?1] about 100 ng PGE₂ on the rat stomach strip. Nonsteroidal anti-inflammatory agents inhibited the release of the contracting substances. Indomethacin, the most potent, was active in concentrations of about 20 ng ml^?1, whereas flufenamic acid was twice, phenylbutazone about 60 times, acetylsalicylic acid 100 times and sodium salicylate about 6000 times less active than indomethacin. The method could prove to be a simple test for screening nonsteroidal anti-inflammatory agents for inhibition of prostaglandin synthesis.     

Inhibition of ACh release by prostaglandin E1 in the rabbit superior cervical ganglion

Superfusion of prostaglandin E₁ (PGE₁) in low concentrations (0.01–0.5 μM) to the neurons of isolated rabbit superior cervical ganglia reversibly depressed the amplitude of the fast excitatory postsynaptic potential (f-epsp) evoked by preganglionic nerve stimulation without changing the resting membrane potential or the membrane resistance. In concentrations higher than 1 μM, PGE₁ elicited a slow membrane depolarization accompanied by a reduction in membrane resistance. PGE₁ in concentrations that did not alter the membrane potential reduced significantly the f-epsp without affecting the acetylcholine (ACh) potential induced by iontophoretic application of ACh to the ganglion cells. The results suggest that the primary action of PGE₁ in the sympathetic ganglia is to depress synaptic transmission by reducing the amount of ACh liberated from preganglionic fibers.     

Metabolism of prostaglandin E2 in the isolated perfused kidney of the rabbit

In the Krebs-perfused rabbit isolated kidney, [³H]PGE₂ (5 μCi, 165 Ci/mmole) was infused intra-arterially for 5 min; venous and urinary effluents were collected every 2 min for 20 min. Efflux of radioactive material peaked at 8 min and declined thereafter. The kidney retained 35% of the infused ³H. Samples were extracted for acidic lipids; PGE₂, PGF_2α and metabolites were separated by TLC and quantified by a radiometric method. Efflux of [³H]PGF_2α into urinary and venous outflows increased progressively over the first 12 min and then plateaued for the remaining 4 min. By 12 min, conversion of [³H]PGE₂ to [³H]PGF_2α was 70 and 80% as determined by radiolabeled products recovered in the urinary and venous effluents respectively. Estimates of total conversion of [³H]PGE₂ to [³H]PGF_2α were 62 and 52% of the radiolabeled material exiting in the urinary and venous effluents respectively. The 15-keto and 13,14-dihydro-15-keto metabolites of [³H]PGF_2α appeared in the urine but were not found in the venous outflow. We conclude that PGE-9-ketoreductase (PGE-9KRD) activity is high in the rabbit isolated perfused kidney. Further, the extent of conversion of PGE₂ to PGF_2α and metabolism of newly formed PGF_2α may differ within the vascular and tubular compartments of the kidney. PGE-9KRD activity may be important in the regulation of renal vascular tone, compliance of veins, and salt and water balance.     

A novel approach to the discovery of non-systemic anti-inflammatory steroids; Antedrug

Therapeutic use of anti-inflammatory steroids is limited due primarily to their systemic suppressive effects on pituitary function and the immune system. To overcome the clinical limitation, a new approach toward the discovery of non-systemic anti-inflammatory steroids is based upon the antedrug concept introduced by this laboratory. The new concept describes locally active agents which are designed to undergo a predictable biotransformation to inactive metabolites upon entry into systemic circulation from the applied site. Thus, true antedrugs are devoid of systemic adverse effects. In a continuing effort, 16alpha-carboxylate and isoxazoline derivatives of prednisolone have been synthesized and screened. In the croton oil-induced ear edema bioassay, the following relative potencies were obtained setting hydrocortisone=1.0; 3a, 1.5; 3b, 3.1; 4a, 4.0; 4b, 12.2; 5b, 8.2; 6b, 11.2; 7a, 1.9; 7b, 4.1; 8a, 3.3; 8b, 6.8; 9a, 0.7; 9b, 8.6; 10a, 2.6; 10b, 7.4. Results of the five-day bioassay indicated that, in contrast to the parent compound, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights, adrenal weights or plasma corticosterone levels. Taken together, the antedrug concept appears to be a fundamentally sound strategy for the separation of local anti-inflammatory activity from systemic adverse effects.