Michal Schwartzman

Prostaglandin generation in rabbit kidney. Hormone-activated selective lipolysis coupled to prostaglandin biosynthesis.

The endogenous release of prostaglandins and free fatty acids from the isolated perfused rabbit kidney in the absence or presence of stimulation by bradykinin or angiotensin-II was investigated. Basal (nonstimulated) release of prostaglandin-precursor arachidonic acid was 15-20-fold higher than that of prostaglandin E2 indicating a low conversion of released arachidonate to prostaglandins. Addition of bovine serum albumin to the perfusion medium caused a substantial (50-250%) increase in the release of all fatty acids except myristic and arachidonic acids, and no significant change in prostaglandin E2 generation. In contrast, administration of bradykinin (0.5 microgram) or angiotensin-II (1 microgram) caused a 10-15-fold increase in prostaglandin E2 release, and with albumin present, also a 2-3-fold selective increase in arachidonic acid release. Thus, unlike what was observed under basal conditions, arachidonic acid released following hormone stimulation is efficiently converted to prostaglandin E2. We conclude that administration of bradykinin or angiotensin-II into the perfused kidney activates a lipase which selectively releases arachidonic acid, probably from a unique lipid entity. This lipase reaction is tightly coupled to a prostaglandin generating system so that the released arachidonate is first made available to the prostaglandin cyclooxygenase, resulting in its substantial conversion to prostaglandins.

Effect of organic sulfur compounds on the chemical and enzymatic transformations of prostaglandin endoperoxide H2

The effects of several sulfur organic compounds on the enzymatic and non-enzymatic transformations of prostaglandin endoperoxide H2 to prostaglandins were studied. Mercaptoethanol, methional alpha-lipoic acid and dimercaptopropanol increased the chemical (i.e. non-enzymatic) reduction of prostaglandin H2 to prostaglandin F2alpha but except for alpha-lipoic acid, had no effect on the enzymatic conversion of prostaglandin H2 to prostaglandin. In contrast, reduced glutathione showed no effect on the chemical conversion of prostaglandin H2, but exerted a marked and specific stimulation on the enzymatic isomerization of prostaglandin H2 to prostaglandin E2. This specific effect of gluthione may serve to regulate the overall intracellular activity of prostaglandin synthetase as well as the particular ratio of prostaglandins produced.

Prostaglandin biosynthesis in rabbit kidney medulla: inhibition in-vitro vs. in-vivo by aspirin, indomethacin and meclofenamic acid.

The non-steroidal anti-inflammatory drugs aspirin, indomethacin and meclofenamic acid were compared for their potency and duration of inhibition of prostaglandin biosynthesis in rabbit kidney medulla. Indomethacin and meclofenamic acid showed equal potency of inhibition in-vitro (IC50 0.88 micron and 0.85 micron respectively) while aspiring was a much weaker inhibitor (IC50 120 micron). In-vivo, indomethacin was the most powerful inhibitor (ID50 0.034 mg/kg) followed by meclofenamic acid (0.45 mg/kg) and aspirin (2.35 mg/kg). Studies on the duration of in-vivo inhibition by these compounds showed the effect of indomethacin and meclofenamic acid to be completely reversed within 4-6 hours. In contrast, return of kidney prostaglandin biosynthetic activity following aspirin inhibition is very slow and significant inhibition is still present 48 hours after a single aspiring injection. The inhibitory effect of aspirin in-vivo could be blocked by pretreatment with indomethacin, indicating that both drugs interact with related sites on the cyclo-oxygenase enzyme. The irreversible inhibition of the cyclo-oxygenase by aspirin as demonstrated in studies of other investigators suggests that the return of kidney prostaglandin synthetase activity after aspirin inhibition represents synthesis of new cyclo-oxygenase protein.

Evidence for different purinergic receptors for ATP and ADP in rabbit kidney and heart

ATP and ADP stimulated the release of specific prostaglandin products from the perfused rabbit kidney heart. The two nucleotides produced the same qualitative profile of prostaglandin products. In kidney, prostaglandin E2 was the major product, whereas in heart 6-keto prostaglandin F1 alpha and prostaglandin E2 predominated. ATP was a slightly more potent than ADP. ATP administered into the perfused heart to kidney was rapidly hydrolyzed to ADP and AMP. The prostaglandin E2 generating activity of ATP was increased 6-10 fold when ATP was given together with AMP-PCP or AMP-PNP which competitively inhibit the activity of vascular ATPase. Thus, the rapid hydrolysis of ATP reduces its agonistic activity for prostaglandin release. ATP and ADP administered together at maximal stimulating doses produced an additive response for prostaglandin E2 release. These results and the results of tachyphylaxis experiments indicate that ATP and ADP interact independently with different types of purinergic receptors.

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

Abstract 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-PGF1α predominates.

Indomethacin but not aspirin inhibits basal and stimulated lipolysis in rabbit kidney.

The concurrent effect of indomethacin or aspirin on prostaglandins (PGs) biosynthesis and on cellular fatty acid efflux were compared. Studies with rabbit kidney medulla slices and with isolated perfused rabbit kidney showed a marked difference between the two non-steroidal anti-inflammatory drugs, with regard to their effects on fatty acid efflux from kidney tissue. While aspirin effect was limited to inhibition of PGs biosynthesis, indomethacin also reduced the release of free fatty acids. In medullary slices, indomethacin inhibited the Ca2+ stimulation of phospholipase A2 activity and the resulting release of arachidonic and linoleic fatty acids. In the isolated perfused rabbit kidney, indomethacin inhibited the basal efflux of all fatty acids as well as the angiotensin II--induced selective release off arachidonate. Indomethacin also blunted the angiotensin II--induced temporal changes in the efflux of all other fatty acids. Neither indomethacin nor aspirin affected significantly the uptake and incorporation of exogenous (14C)-arachidonic acid into kidney total lipid fraction. Our tentative conclusion is that indomethacin inhibits basal as well as Ca2+ or hormone stimulated activity of kidney lipolytic enzymes. This action of indomethacin reduces the pool size of free arachidonate available for conversion to oxygenated products (both prostaglandin and non-prostaglandin types). The non-steroidal anti-inflammatory drugs can therefore be divided into two groups: a) aspirin-type compounds which inhibits PGs formation only by interacting with the prostaglandin endoperoxide synthetase and b) indomethacin-type compounds which inhibit PG generation by both reduction in the amount of available arachidonate and direct interaction with the enzyme.

Selective induction of de novo prostaglandin biosynthesis in rabbit kidney cortex

Ureter-obstructed kidney develops during perfusion an enhanced responsiveness to bradykinin-stimulated prostaglandin release. This enhanced prostaglandin generation results from de novo synthesis of prostaglandin synthetase and acylhydrolase enzymes during the perfusion and is therefore unaffected by acetylsalicylic acid (aspirin) inhibition of prostaglandin synthesis prior to initiation of perfusion. Studies were carried out to identify the renal cellular site in which the newly synthesizing prostaglandin generating system is localized. Kidneys with or without aspirin treatment were perfused for either 1 or 5 h. Following perfusion, medullary and cortical slices were incubated and prostaglandin E2 production measured. Medullary slices showed similar prostaglandin E2 biosynthetic activity in kidneys perfused for 1 or 5 h. Furthermore, medullary prostaglandin generation was inhibited (90-95%) by aspirin pre-treatment and did not increase during subsequent perfusion for 5 h. In contrast, cortical slices from kidneys pretreated with aspirin regained their full activity after 5 h or perfusion, this regeneration being abolished by infusion of the protein synthesis inhibitor, cycloheximide. The same differences in activities between medulla and cortex were also seen when microsomal fractions were compared. The perfusion-induced formation of prostaglandin synthetase activity is thus specifically localized in the cortex and can be detected in cortical microsomes. This cortical activity is unique in that endogenous arachidonic acid released from esterified lipids is converted to prostaglandins, whereas exogenous added arachidonic acid is not. It thus appears that the induced cortical acylhydrolase and prostaglandin synthetase activities are tightly coupled and that the true molecular form or precursor arachidonate for this prostaglandin generating system is esterified and not free arachidonate.

Bradykinin-stimulated differential incorporation of arachidonic acid into lipids of kidney cortex and medulla☆

We investigated bradykinin-induced changes in the turnover of arachidonate in renal lipids of the perfused rabbit kidney. Upon hormone stimulation, this cellular system undergoes only transient dynamic changes in arachidonic acid metabolism; no loss of bradykinin effect on arachidonate release and prostaglandin generation is shown upon repeated hormone administrations during 8-9 hr of perfusion. Ureter-obstructed rabbit kidneys were perfused for 5-6 hr and then saline or bradykinin in saline was administered, followed after 10 sec by pulse labelling with [14C]arachidonate. The pattern of distribution of [14C]arachidonate in lipid fractions of the cortex showed that bradykinin caused a 2 to 2.5-fold increase in the relative incorporation of arachidonic acid into phosphatidylinositol (PI), phosphatidic acid (PA), diglyceride (DG) and triglyceride (TG) fractions and a concomitant decrease in its incorporation into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In contrast, in the medulla hormone administration caused a marked increase of arachidonate incorporation into PI and PC, and a decrease in incorporation into PE, PA, DG and TG. This differential arachidonate labelling of cortical vs medullary lipids following bradykinin stimulation suggests that the hormone activates different lipolytic processes in cortex and medulla, and promotes hydrolysis of arachidonic acid from different phospholipid pools.

Distinct Acylhydrolase and PG Synthase Systems in the Perfused Rabbit Kidney

The endogenous generation of prostaglandins (PGs) and other oxygenated products of arachidonic acid is a complex process which involves acylhydrolase action to generate unesterified arachidonic acid and subsequent conversion to cell-specific oxygenated products. Control of this process can occur at several key steps, including the following: (1) the cellular site for agonist recognition and interaction, (2) the lipolytic step, (3) the extent of metabolic coupling between the released arachidonate and its transformation to oxygenated products, (4) the types of arachidonate metabolizing enzymes in the particular tissue or cell affected, and (5) other related reactions. In this report we employ the ureter-obstructed kidney model for the comparative study of the lipolytic and PG synthetic mechanism which are stimulated by the peptide hormones bradykinin and angiotensin II, the adenine nucleotides ATP and ADP, and by exogenous administration of unesterified arachidonic acid.

Perfusion-Dependent Selective Induction of Prostaglandin Biosynthesis Activity in Rabbit Kidney Cortex

Renal prostaglandin release, induced by peptide hormones, involves a selective deacylation of esterified arachidonic acid in a tightly coupled process in which a major portion (25–50%) of the released arachidonate is converted to prostaglandins.1,2 In the ureter-obstructed kidney model employed in many of these studies, there is a time-dependent increase in prostaglandin release in response to bradykinin or angiotensin II stimulation. This enhanced prostaglandin release is the result of a de novo gradual synthesis of both a hormone-sensitive lipase and prostaglandin synthase enzymes during the perfusion.2,3 A major question yet to be resolved concerns the renal cellular site in which the newly synthesizing prostaglandin synthase activity is located. This report provides evidence that the newly synthesized prostaglandin synthase is found in the cortex of the perfused, ureter-obstructed kidney.