Stimulation of Prostaglandin Biosynthesis by Adenine Nucleotides

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1 Stimulation of Prostaglandin Biosynthesis by Adenine Nucleotides PROFILE OF PROSTAGLANDIN RELEASE BY PERFUSED ORGANS SyPhilip Needleman, Mark S. Minkes, and James R. Douglas, Jr. ABSTRACT Prostaglandin release from various isolated perfused organs is characteristically dependent on the stimulus; thus, certain stimuli caused prostaglandin release in some organs and not in others. Adenosine triphosphate and adenosine diphosphate were potent stimulators of prostaglandin biosynthesis in the kidney, spleen, spleen fat pad, heart, liver and lung; adenosine monophosphate and adenosine were inactive. Epinephrine caused prostaglandin release from the kidney, spleen, and liver, whereas, angiotensin was agonistic in the kidney, spleen, spleen fat pad, and liver. Indomethacin abolished the release (biosynthesis) of prostaglandins from all organs by each agonist. KEYWORDS indomethacin rabbit rat heart fat pad lung rat stomach strip autoregulation adenosine triphosphate liver chick rectum A variety of smooth muscle preparations respond to adenine nucleotides. The predominant response of mammalian intestinal smooth muscle to adenosine triphosphate (ATP) is relaxation (l). Adenyl compounds dilate most blood vessels (2), but they constrict pulmonary vessels (3) and isolated renal arteries (4). Burnstock (5) has reported that ATP is the transmitter released from nonadrenergic inhibitory neurons in the gut, and he has proposed classifying such neurons as purinergic nerves. Dipyridamole potentiates the effects of both exogenously applied ATP and stimulation of nonadrenergic (purinergic) inhibitory fibers in guinea pig taenia coli (6), whereas quinidine antagonizes these actions (l). In the current study, ATP and adenosine diphosphate (ADP) acted as potent releasers of a prostaglandinlike substance from a wide variety of isolated perfused organs. These organs did not store prostaglandin; thus, release was a reflection of de novo synthesis (7). However, the ability of the adenine nucleotides to release prostaglandin appeared to be unaffected by dipyridamole or quinidine (modifiers of purinergic neurotransmitter responses). From the Department of Pharmacology, Washington University Medical School, St. Louis, Missouri This work was supported by U. S. Public Health Service Grants HE , HE-11771, and GM from the National Institutes of Health. A preliminary report of this work was presented at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, August 23, 1973, East Lansing, Michigan (Pharmacologist 15:250, 1973). Received May 4, Accepted for publication January 11, Circulation Research. VoL XXXIV, April 1974 RABBIT KIONEY PERFUSION Methods New Zealand rabbits (females) weighing kg were anesthetized with sodium pentobarbital (30 mg/kg, iv). After the abdominal cavity had been opened, each rabbit was heparinized (250 units/kg), and polyethylene catheters were inserted and tied into the renal artery (PE 160, Clay Adams), the renal vein (PE 205), and the ureter (PE 90). The kidney was placed in a warming jacket and perfused with oxygenated (95% O 2-5% CO 2 ) Krebs-Henseleit solution at 37 C. The flow rate was held constant (8 ml/min); therefore, changes in perfusion pressure were indicative of alterations in vascular resistance. Perfusion pressure was measured with a P-1000A linear-core pressure transducer (Physiograph). Renal ischemia was achieved by temporarily diverting (with a three-way stopcock) the flow of perfusion fluid away from the renal artery and directing it onto the assay organs. RABBIT SPLEEN PERFUSION After the rabbit was anesthetized, the abdomen was opened and the gastrosplenic blood vessels were ligated and cut. Mesenteric fat was partially dissected away from the spleen. The rabbit was then heparinized (250 units/kg, iv), and the splenic artery was cannulated (PE 50, Clay Adams). The spleen was removed from the rabbit, and perfusion was begun with a constant flow of Krebs solution (6 ml/min, 95% O 2-5% CO 2 ) at 37 C. Changes in vascular resistance were recorded as described for the kidney. RABBIT SPLEEN FAT PAD PERFUSION Splenic fat pads were prepared similarly except that the vascular branches into the spleen were ligated and the spleen dissected away. The spleen fat pad was perfused with Krebs solution at 5 ml/min; uniform perfusion 455

2 / - / / 456 NEEDLEMAN, MINKES, DOUGLAS of the entire fat pad was confirmed with a fluorescent dye (sodium fluorescein). RABBIT HEART PERFUSION The heart was removed from the anesthetized rabbit, and the aorta was cannulated just superior to the aortic valves with a blunted 13-gauge needle. The beating heart was perfused at a constant flow rate (10 ml/min) with oxygenated (95% O 2-5% CO 2, 37 C) Krebs solution so that changes in perfusion pressure were indicative of alterations in vascular resistance contributed by the coronary vessels. RAT LIVER PERFUSION Krebs solution was pumped into the rat liver at 16 ml/ min through the portal vein, and the effluent emerged from a vena cava cannula and cascaded across the assay organs. RAT LUNG PERFUSION The pulmonary artery was cannulated through the right ventricle and perfused with Krebs solution at 4 ml/ min through the air-inflated lungs. The effluent from the pulmonary veins cascaded across the assay organs. An additional 4 ml/min of Krebs solution was delivered across the assay organs, which were therefore bathed with 8 ml/min of media. SUPERFUSED ORGAN SYSTEMS Isolated assay tissues (rat stomach strip and chick rectum) were superfused by the venous effluent from the isolated perfused organs (kidney, spleen, heart, etc.), according to the procedure of Vane (8). The rat stomach strip and the chick rectum are particularly sensitive to prostaglandins. That the substances detected were most likely prostaglandins is based primarily on the specificity of the systems used for bioassay (9) but is reinforced by the disappearance of activity after treatment with indomethacin, a specific inhibitor of prostaglandin biosynthesis (10). In addition, in the case of the splenic venous effluents, we had the opportunity to specifically verify the presence of prostaglandin in the E type by radioimmunoassay, and the results paralleled the response obtained with the specific assay organs and with the indomethacin inhibition (11). Rat colon strips were also present in the superfusion, but we obtained no evidence for PGF 2(, release by any of the stimuli tested. A mixture of antagonists was added to the superfusion fluid at 0.1 ml/min; they rendered the assay tissues insensitive to catecholamines, acetylcholine, serotonin, and histamine (8). The antagonist reagent consisted of Krebs solution containing 1 mg of phenoxybenzamine, 1 20 mg of propranolol, 2 1 mg of hyoscine hydrobromide,2 mg of methysergide, 3 and 1 mg of diphenhydramine per 'Kindly supplied by Smith, Kline, and French. 2 Kindly supplied by Ayerst. 3 Kindly supplied by Sandoz. 100 ml. Indomethacin 4 (10 jug/min) was also superfused directly over the assay organs to eliminate the possibility that they released prostaglandin and also to enhance their sensitivity to prostaglandin (12). Since indomethacin only has a limited aqueous solubility, a stock solution (10 mg/ml) was prepared in 95% ethanol. The stock solution was then diluted to 100 Mg/ml in Krebs- Henseleit solution (ph 7.4). This solution was kept on ice and perfused at 0.1 ml/min into the Krebs solution as it entered the perfused organs. Fresh aqueous indomethacin solutions were prepared every 3 hours. Substances were tested (as bolus injections) for their effect on the assay organs by adding the agent to the CHICK RECTUM PGE, E, E, 5ng long 2Ong DIR DIR DIR 20 RAT STOMACH 40 ng DIR 20 x!0'' moles ATP TK. >39ng /,V5xlO' / ATP mj PGE, 7 ' moles TK CHICK RECTUM 20xlO''rr ATP TK ATP 20x10'' moles TK otes x / x^5 x 10"'moles / ATP TK '/ I8ng / ng PGE2 - ATP 20x10' moles DIR I 203 I FIGURE 1 Dose-response curve of rat stomach strip and chick rectum to prostaglandin 2 (PGE2) standards and to A TP-induced release of prostaglandinlike substance (). The top two curves illustrate an experiment in which increasing concentrations of PGE2 were added directly (DIR) to the Krebs-Henseleit solution as it superfused the assay organs. TK indicates that the A TP was injected through the kidney. Following infusion of indomethacin (10 ixg/min) into the kidney, the response to an injection of ATP througli the kidney was indistinguishable from the response produced by applying ATP directly to the assay tissues (not shown). The bottom two graphs show the plots obtained from the contractile data. The closed circles indicate the PGE2 standards, and the crosses represent the prostaglandinlike substance in the renal venous effluent following ATP injection through the kidney. " Kindly supplied by Merck, Sharp, and Dohme. Circulation Research, Vol. XXXIV, April 19T4

3 PROSTAGLANDIN RELEASE BY ATP 457 perfusion fluid after it had passed through the perfused organ. The response of the perfused organ to the drug was determined by injecting the agent into the Krebs solution by way of an injection port just proximal to the arterial cannula. Changes in smooth muscle tension were measured with F-50 microdisplacement myographs (Physiographs). The method used to calculate the quantity of prostaglandinlike substance released from the perfused organs by various agonists is illustrated in Figure 1. These data were obtained from a kidney perfusion experiment in which the renal venous effluent superfused a rat stomach strip, a chick rectum, and a rat colon. Rat colon responses are not shown, since neither the PGE 2 standards nor the various agonists (direct or through the kidney) contracted the tissue. The contractile responses of a chick rectum and a rat stomach strip to increasing doses of PGE 2 applied as a bolus directly over the assay organs are compared with the responses induced by release of a prostaglandinlike substance by ATP injected as a bolus through the kidney (top two records, Fig. 1). The contractile response of each tissue was then plotted (Fig. 1) to facilitate comparison of ATP-induced release of prostaglandinlike substance to PGE 2 standards. The response of rat stomach strips to released prostaglandinlike substance was corrected for the contraction produced by ATP when it was directly tested over the assay organs. Indomethacin, which blocks prostaglandin biosynthesis, abolished renal release of prostaglandinlike substance but did not decrease the response to PGE 2 standards applied directly over the assay organs (not shown). In each experiment the assay organs were calibrated with prostaglandin 5 standards which were tested at the beginning and the end of the experiment, and the response to prostaglandinlike substance Renal Prostaglandin Release by Isolated Perfused Rabbit Kidneys Renal pretreatment None (8) Dipyridamole, 3 /xg/ml min~' (4) Quinidine, 8 /^g/ml min" 1 (5) Indomethacin, 1.3 fig/m\ min" 1 (8) APP (mm Hg) 9 ± 3 12 ± 5 14 ± 7 10 ± 2 TABLE 1 ATP released from the perfused organ was compared with that to the standards; thus oath experiment served as its own control. RENAL PROSTAGLANDIN RELEASE Results When ATP was injected into the kidney, the perfusion pressure increased slightly and there was an immediate release of prostaglandinlike substance as evidenced by contraction of the rat stomach strip and the chick rectum. The ATP-induced release of prostaglandinlike substance was calculated to be 24 ± 5 ng (N = 8, Table 1 from PGE 2 dose-response curves) (as illustrated in Fig. 1). Epinephrine caused a marked increase in perfusion pressure and a distinct release of prostaglandinlike substance (19 ± 4 ng. Table 1), when it was injected through the kidney. The superfused assay organs were constantly exposed to indomethacin; therefore, release of prostaglandinlike substance could only arise from the perfused rabbit kidney. In addition, the assay organs were constantly exposed to adrenergic blocking agents (both alpha- and beta-receptor blockers); therefore, epinephrine could not exert any direct effects on the assay tissues. Rat colon strips, which are sensitive to PGF 2a showed little or no response to renal venous effluent from rabbit kidneys treated with either ATP or epinephrine. Preinfusion of the kidneys with either dipyridamoile or quinidine did not markedly alter the release of prostaglandinlike (ng) 24 ± 5 27 ±9 21 ±7 0 APP (mm Hg) 52 ± 4 58 ± ± 6 65 ± 6 Epinephrine (ng) 19 ±4 17 ±7 3 ±2 0 All values are means ± SE. ATP (5X10 7 moles) and epinephrine (5x10 7 moles) were administered as bolus injections (50 ^liters) into the Krebs-Henseleit solution as it entered the kidney. The pretreatments were continuously infused into the kidney for at least 15 minutes before challenging with either ATP or epinephrine. In any given experiment, the agonists were tried alone as well as with one of the pretreatment infusions. APP = change in perfusion pressure, and = prostaglandinlike substance appearing in the renal venous effluent calculated from contractions of the chick rectum and the rat stomach strip compared with contractions of these organs induced by standard prostaglandin as previously described (13). Numbers in parentheses indicate the number of isolated rabbit kidneys tested. 5 Kindly supplied by Upjohn. Circulation Research, Vol. XXXIV. April 1974

4 458 NEEDLEMAN, MINKES, DOUGLAS substance by ATP. Quinidine decreased the renal constriction and the release of prostaglandinlike substance induced by epinephrine; however, the quinidine response was completely reversible. Indomethacin, which blocks prostaglandin biosynthesis (10), abolished ATP- or epinephrine-induced release of prostaglandinlike substance (Table 1). PROFILE OF PROSTAGLANDIN RELEASE FROM PERFUSED ORGANS ATP and ADP (5 X 10" 7 to 5 X 10~ 6 moles) were potent stimulators of prostaglandin biosynthesis in a wide variety of organs perfused with Krebs solution including the rabbit spleen, rabbit kidney, rabbit heart, rabbit spleen fat pad, rat liver, and rat lungs (Table 2). In each case, the release of the prostaglandinlike substance was completely blocked by indomethacin. ATP and ADP were the only compounds tested that released prostaglandinlike substance from all these organs. 5'- Adenosine monophosphate, adenosine, cyclic 3', 5'- adenosine monophosphate, and guanosine triphosphate were inactive as releasers of prostaglandinlike substance at one hundred times the effective molar concentration of ATP. Some restraints arise when the release of prostaglandinlike substance from isolated perfused rat livers or rat lungs is considered. These two organs elicited a greater variability than that which occurred with other tissues, and there were occasional preparations in which there was no release of prostaglandinlike substance regardless of the stimulus. The lungs released prostaglandinlike TABLE 2 Profile of Prostaglandin Release from Isolated Perfused Organs Stimuli Kidney Spleen ATP, (5 x 10~ 8 moles) ADP (5 X 10~ 8 moles) Adenosine (10~ 5 moles) Ischemia (4 minutes) Epinephrine (5 X 10~ 7 moles) Angiotensin (10~ 10 moles substance when ATP was injected into the Krebs solution as it entered the pulmonary artery. The only other stimulus which caused release of prostaglandinlike substance in the lung experiments was inflating and deflating the lungs several times. Angiotensin II stimulated release of prostaglandinlike substance from the perfused kidney, spleen, spleen fat pad, and liver (Table 2). Angiotensin has also been demonstrated to release prostaglandinlike substance from arteriolar vascular smooth muscle of the perfused rabbit mesenteric bed (14). However, angiotensin does not release prostaglandinlike substance from the coronary or the pulmonary circulation (Table 2) or from skeletal muscle or cutaneous vasculature (measuring the appearance of prostaglandinlike substance in dog femoral venous blood following angiotensin infusion into the femoral artery [15]). Ischemia was not a universal stimulus for release of prostaglandinlike substance from perfused organs (Table 2). Adenosine has been implicated in autoregulation, but in these experiments even high concentrations (up to 10~ 5 moles) did not enhance prostaglandin biosynthesis. Discussion It has been proposed that purine nucleotides have a physiological role in the regulation of blood flow in the coronary arteries (16, 17). Adenosine appears in coronary sinus blood during reactive hyperemia in the dog heart (18). Recently, Her- Spleen fat pad Heart Liver Lung ± ++ The positive (+) results indicate the release of prostaglandin in amounts (10 ng or more) which can readily be detected with the superfused assay tissues. The experiments were performed separately with perfused organs from at least five animals. Negative ( ) results indicate no detectable prostaglandin release, whereas ± indicates variable results. The stimulation conditions are those listed in the table with the following exceptions. The stimulus parameters required in heart were: ATP or ADP 5 X 10~ 7 moles, ischemia 8 minutes (5 out of 11 preparations released prostaglandinlike substance, but no prostaglandin release was induced by angiotensin (up to 10~ 7 moles) or epinephrine (up to 5 x 10~ 6 moles). The spleen fat pad required: ATP or ADP 5 X 10~ 7 moles, angiotensin 10~ 9 moles; epinephrine induced no response at 5 x 10~ 6 moles. Rat liver required: ATP 10~ 6 moles, epinephrine 2 x 10~ 6 moles', angiotensin 5 x 10~ 9 moles, and ischemia 8 minutes (only 4 out of 7 preparations responded with prostaglandin release). Rat lungs responded only to ATP 10~ 6 moles. Circulation Research, VoL XXXIV, April 1974

5 PROSTAGLANDIN RELEASE BY ATP 459 bacyzynska-cedro and Vane (19) have demonstrated that autoregulation in the kidney is associated with elevated prostaglandin levels and that renal reactive hyperemia is abolished by indomethacin. The current investigation demonstrated that adenine nucleotides were potent stimulators of prostaglandin release in a number of critical organs. Recently, preliminary data have been presented indicating that PGE and PGA are released from the coronary circulation of the dog during postocclusion reactive hyperemia (20). We demonstrated that ischemia or ATP caused prostaglandin release from isolated perfused rabbit hearts and that indomethacin abolished the ATPinduced coronary prostaglandin release and markedly decreased the ATP-induced coronary vasodilation (21). The injection of PGE, into Krebsperfused isolated guinea pig hearts results in increased coronary flow and a fourfold increase in the concentration of adenosine in the perfusate (22). The possibility emerges that these agents (i.e., adenine nucleotides, adenosine, and prostaglandins) working in concert are the controlling influences in autoregulation. Some recent reports have associated adenine nucleotides with prostaglandin release. Platelet prostaglandin synthesis may be involved in ADPinduced aggregation (23). ADP has also been shown to enhance the formation of PGE, in rat brain homogenates, but ATP inhibits the formation of PGE 3 (24). Malik and McGiff (25) have demonstrated that ATP and ADP inhibit adrenergic transmission in isolated perfused rat mesenteric arteries. Their data can possibly be explained by our demonstration that adenine nucleotides enhance prostaglandin biosynthesis (Table 2), since prostaglandins have been demonstrated to inhibit the release of norepinephrine from adrenergic nerve terminals (26). ATP and ADP may prove to be useful tools in establishing the ability of an organ, a tissue culture, or a body region to synthesize and release prostaglandins. Acknowledgment The authors are grateful for technical assistance from Mrs. Patricia B. Stoecklein and Mrs. Anne H. Kauffman. References 1. BURNSTOCK. C, CAMPBELL, G., STACHELL, D. C, AND SMYTHE, A.: Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by nonadrenergic inhibitory nerves in the gut. Br J Circulation Reiearch, Vol. XXXIV, April 1974 Pharmacol 40: , HADDY, F.J., AND SCOTT, J.B.: Metabolically linked vasoactive chemicals in local regulation of blood flow. Physiol Rev 48: , EMMELIN, N., AND FELDBERC, W.: Systemic effects of * adenosine triphosphate. Br J Pharmacol 3: , HRDINA. P.D., BONACCORSI, A., AND GARATTINI, S.: Pharmacological studies on isolated and perfused rat renal arteries. Eur J Pharmacol 1:99-108, BURNSTOCK, G.: Purinergic nerves. Pharmacol Rev 24: , STACHELL, D.G., LYNCH, A., BOURKE, P.M., AND BURNSTOCK, G.: Potentiation of the effects of exogenously applied ATP and purinergic nerve stimulation on the guinea pig taenia coli by dipyridamole and hexabendine. Eur J Pharmacol 19: , PIPER, P.J., AND VANE, J.R.: Release of prostaglandins from lung and other tissues. Ann NY Acad Sci 180: , VANE, J.R.: Use of isolated organs for detecting substances in the circulating blood. Br J Pharmacol 23: , GILMORE, N., VANE, J.R., AND WYLLIE, J.H.: Prostaglandin released by the spleen. Nature (Lond) 218: , VANE.J.R.: Inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs. Nature [New Biol] 231: , DOUGLAS. J.R., JR., JOHNSON, E.M., JR., MARSHALL, G.R., JAFFE, B.M., AND NEEDLEMAN, P.: Stimulation of splenic prostaglandin released by angiotensin and specific inhibition by cysteine 8 -AII. Prostaglandins 3:67-74, 12. ECKENFELS, A., AND VANE, J.R.: Prostaglandins, oxygen tension and smooth muscle tone. Br J Pharmacol 45: , NEEDLEMAN P., KAUFFMAN, A.H., DOUGLAS, J.R., JR., JOHNSON, E.M., JR., AND MARSHALL, G.R.: Specific stimulation and inhibition of renal prostaglandin release by angiotensin analogs. Am J Physiol 244: , 14. NEEDLEMAN, P., MARSHALL, G.R., AND DOUCLAS, J.R., JR.: Prostaglandin release from vasculature by angiotensin II: Dissociation from lipolysis. Eur J Pharmacol 66: , 15. AlKEN, J.W., AND VANE. J.R.: Intrarenal prostaglandin release attenuates the renal vasoconstrictor activity of angiotensin. J Pharmacol ExpTher 184: , 16. BERNE, R.M.: Regulation of coronary flow. Physiol Rev 44:1-29, BERNE. R.M., RUBIO, R., DOBSON, J.D., AND CURNISH, R.R.: Adenosine and adenine nucleotides as possible mediators of cardiac and skeletal muscle blood flow regulation. Circ Res 28(suppl. I):I , RUBIO, R., BERNE, R.M., AND KATORI, M.: Release of adenosine in reactive hyperemia of the dog heart. Am J Physiol 216:56-62, HERBACYZYNSKA-CEDRO, K., AND VANE, J.R.: Contribution of intrarenal generation of prostaglandin to autoregulation of renal blood flow in the dog. Circ Res 33: , 20. KRAEMER, R.J., AND FOLTS, J.D.: Release of prostaglandin

6 460 NEEDLEMAN, MINKES, DOUGLAS following temporary occlusion of the coronary artery 24. (abstr.). Fed Proc 32:454, 21. MlNKES, M, AND NEEDLEMAN, P.: Prostaglandin release by the isolated perfused rabbit heart. Prostaglandins 25. 3: , 22. LOCAN, ME., AND WlEDMEIER, V.T.: Changes in myocardial adenosine concentrations during prostaglandin-induced coronary vasodilation (abstr.). Fed Proc 32:3221, SHIO, H., AND RAMWELL, P.: Effect of prostaglandin E 2 and aspirin on the secondary aggregation of human platelets. Nature [New Biol] 236:45-46,1972. ABDULL, Y.H., AND MCFARLAND, E.: Control of prostaglandin biosynthesis in rat brain homogenates by adenine nucleotides. Biochem Pharmacol 21: , MALIK, K.U., AND MCGIFF, J.C.: Modulation by carbohydrates and their metabolites of adrenergic transmission in rat mesenteric arteries (abstr.). Fed Proc 32:706, STJARNE, L.: Alpha-adrenoceptor mediated feed-back control of sympathetic neurotransmitter secretion in guinea pig vas deferens. Nature [New Biol] 241: , Circulation Research. VoL XXXIV, April 1974