233
Journal of Physiology (1990). 428. pp. 233-241 W'ith 5 fig res Prinited in Great Britain
EFFECT OF MELITTIN ON RENIN AND PROSTAGLANDIN E2 RELEASE FROM RAT RENAL CORTICAL SLICES
BY PAUL C. CHURCHILL, NOREEN F. ROSSI, MONIQUE C. CHURCHILL AND VIRGINIA R. ELLIS From the Department of Physiology and Internal Medicine, Wayne State University School of Medicine, Detroit, MIU 48201, UTSA
(Received 16 February 1990) SUMMARY
1. The present experiments were designed to determine the effect of melittin on renin secretion. Melittin is a polypeptide component of bee venom which stimulates phospholipase A2 activity, thereby increasing arachidonic acid release and prostaglandin (PG) synthesis, and which inhibits protein kinase C activity. Either of these actions might be expected to stimulate renin secretion, since renin secretion is stimulated by arachidonic acid and by several PGs, and since renin secretion is inhibited by several activators of protein kinase C. 2. In rat renin cortical slices incubated at 37 'C in a buffered and oxygenated physiological saline solution, 0 -0l uM-melittin produced a concentration-dependent stimulation of both prostaglandin E2 (PGE2) synthesis and renin secretion. However, melittin-stimulated renin secretion is independent of melittin-stimulated phospholipase A2 activity, arachidonic acid release, and PG synthesis, since 20 ,umquinacrine (a phospholipase A2 antagonist) and 50 /tM-meclofenamate (a cyclooxygenase antagonist) antagonized basal and melittin-stimulated PGE2 synthesis but had no effects on basal or melittin-stimulated renin secretion. 3. Furthermore, melittin-stimulated renin secretion is not produced by inhibition of protein kinase C, since an activator of protein kinase C (12-O-tetradecanoylphorbol 13-acetate, TPA), enhanced rather than antagonized melittin-stimulated renin secretion. Ouabain partially antagonized, but did not completely block, melittinstimulated renin secretion. 4. Thus, melittin-stimulated phospholipase A2 activity probably accounts for stimulated PGE2 production, but not for stimulated renin secretion. The mechanism of melittin-stimulated renin secretion is unclear; an effect on protein kinase C does not appear to be involved, and in contrast to the stimulatory effects of a variety of other substances, melittin-stimulated renin secretion is only partially antagonized by ouabain. INTRODUCTION
Both the renal cortex and the renal medulla synthesize PGs (Sun, Taylor, McGuire & Wong, 1981), and several investigators have suggested that endogenous PGs play a role in the physiological control of renin secretion (Beierwaltes, Schryver, .NIS 8281
234
P. C CHURCHILL AND OTHERS
Olson & Romero, 1980; Keeton & Campbell, 1980; Henrich, 1981; Beierwaltes,
Schryver, Sanders, Strand & Romero, 1982; Freeman, Davis & Villarreal. 1984). Three lines of evidence support this suggestion (reviewed by Henrich, 1981). First, many exogenous (PGs have been shown to stimulate renin secretion in vivo, and in a wide variety of in vitro preparations including isolated glomeruli (Beierwaltes et al. 1982). Effects in the latter preparation demonstrate that the effects are exerted directly, independently of PG-induced changes in renal haemodynamics and/or the renal tubular handling of NaCl. Second, exogenous arachidonic acid stimulates renin secretion in vivo and Hin vitro, and this response is mediated by newly synthesized PGs. since it is antagonized by cyclo-oxygenase inhibitors. Third, cyclo-oxygenase inhibitors have been shown to suppress both basal and stimulated renin secretion in vivo. Collectively, these observations have led some investigators to hypothesize that PG synthesis mediates renin secretion (Henrich, 1981). The present experiments were designed to further explore the relationship between renin secretion and PG synthesis using melittin as a probe. Melittin is a polypeptide component of bee venom which, in all cells studied to date, activates phospholipase A2 activity, thereby increasing arachidonic acid release and PG synthesis (Hassid & Levine, 1977; Argiolas & Pisano, 1983; Tanaka, Kagawa, Murakoso, Shimizu & Matsuoka, 1983). Although there are no previous reports of its effects on renin secretion per se, melittin has been shown to release membrane-bound renin (Nishimura, Alhenc-Gelas, White & Erdos, 1980; Nishimura, XWard & Erdos, 1980), and to stimulate the release of other stored secretory products (Heisler, Reisine, Hook & Axelrod, 1982; Tanaka et al. 1983), observations which suggest that it might stimulate both renin secretion and PG synthesis. Also, melittin might be expected to stimulate renin secretion by another mechanism, independently of phospholipase A2 activity, arachidonic acid release, and PG synthesis: melittin inhibits protein kinase C activity (Nishizuka, 1984), and several observations suggest an inverse relationship between renin secretion and protein kinase C activity (Churchill & Churchill, 1984; Kurtz, Pfeilschifter, Hutter, Buhrle, Nobiling, Taugner, Hackenthal & Bauer, 1986). Therefore, experiments were designed to determine the effect, and the mechanism of action, of melittin on renin secretion. METHODS
The procedures for studying renin and PGE2 release using rat renal cortical slices have been described in detail previously (Churchill. 1979; Churchill, Savoy-MNoore & Churchill. 1983). For each of several experiments, five ether-anaesthetized adult male Sprague-Dawley rats acted as kidney donors. After bilateral nephrectomy, the rats were killed with ether and bilateral pneumothorax. The renal capsule was removed from each kidney. and using a razor blade, four thin slices were cut. The resulting forty slices were distributed between incubation flasks (two slices per flask) each of which contained 12 ml of incubation medium at 37 °C, previously equilibrated with a mixture of 02 (95%) and CO2 (5%). The flasks were stoppered and placed in a oscillating water bath maintained at 37 'C. The gas phase in each flask was continuously replaced throughout the incubation period. Samples of media (1 ml) were taken at 30. 60 and 90 min. The samples were centrifuged (4 'C; 8000 g; 5 min) and the supernatants were stored at -70 'C until analysed for renin and PGE2. After the incubations. tissue dry weight was determined. Dry weights ranged between 0-58 and 6-40 mg per flask (two slices) and averaged 2-95+0-08 mg (n = 318 flasks). The composition of the control incubation medium was: NaCl, 125 mM; NaHCO3 19 mm; KCl, 4 mM; CaCl2, 2-5 mm; NaH2PO4, 1-2 mm; MgSO4. 08 mM; and 0-2 g (100 ml)-' each of glucose and bovine serum albumin. Dose-response curves were generated using melittin concentrations ranging
RENIN AND PROSTAGLANDIN E2 RELEASE
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from 01 to 10 /IM. Meclofenamate, ouabain (Strophanthin G), quinacrine, and 12-0-tetradecanoylphorbol 13-acetate (TPA) were used in some experiments at concentrations which were shown in previous experiments to produce maximal effects (Churchill et al. 1983; Churchill & Churchill, 1984; Churchill, Churchill & McDonald, 1985). A set of control slices was run simultaneously with experimental slices. Renin concentration of the incubation medium was determined using a modification (Churchill et al. 1983) of a previously described method (Churchill, 1979). In brief, renin standards and renincontaining supernatants were incubated with rat renin substrate at 37 °C for 30 min. The angiotensin I generated during this incubation was determined by radioimmunoassay. Renin concentration was calculated as nanograms angiotensin I generated per millilitre of renincontaining sample per hour of incubation with renin substrate (ng ml-' h-'). There was a linear relationship between the renin concentration of the standards expressed as ng ml-' h-' and expressed as Goldblatt Units per millilitre (GU ml-'). This relationship was used to convert renin concentrations of the unknowns from ng ml-' h-' to GU ml-'. The total renin secreted at a given time during the incubation of the slices was calculated as the renin concentration of the medium (GU ml-') multiplied by the volume of the medium at the time of sampling (e.g. 12 ml at 30 min, etc.) and divided by tissue dry weight, yielding the units GU g-'. Renin secretory rate was calculated as the increment in the total amount of renin during the final hour of incubation (from 30 to 90 min), and expressed as GU g-' (60 min)-'. The first 30 min period was taken as an
equilibration period. A melittin-free sample of renin-containing incubation medium was divided into two equal portions. Melittin was added to one portion (1O IM final concentration), and both portions were placed in the 37 °C water bath for 90 min to simulate the conditions for incubating kidney slices. Then, six aliquots of each portion were incubated with renin substrate as described above, and angiotensin I in each of these was determined in duplicate by radioimmunoassay. Renin concentrations of melittin-free and melittin-containing samples averaged 20-4 ±1-4 and 16-9 + 1-5 ng ml-' h-'. respectively. These means do not differ significantly (P > 0 1). Therefore, melittin neither activates an inactive form of renin nor affects the reaction between renin and renin substrate. PGE2 in supernatants was measured using a commercial radioimmunoassay kit (New England Nuclear) without any preprocessing, as discussed previously (Churchill et al. 1983). The total amount released by the slices was calculated as the concentration in the medium (ng ml-'), multiplied by the volume of the medium at the time of sampling (e.g. 12 ml at 30 min, etc.), and divided by tissue dry weight (g) yielding the units ng g-'. Release rate was calculated as the increment in ng g-' during the final hour of incubation, and expressed in the units ng g-' (60 min)-'. All results are expressed as means + S.E.M.s. Analysis of variance and Scheffe contrasts (Wallenstein, Zucker & Fleiss, 1980) were used to assess the statistical significance of differences between means. P values < 0-05 were considered statistically significant. RESULTS
Control or basal renin secretory rate averaged 106 +0-4 GU g-1 (60 min)-' (n = 54). As can be seen in Fig. 1, melittin stimulated renin secretory rate in a concentration-dependent manner. Each point on the dose-response curve differs significantly from the control or basal secretory rate, shown as the filled bar. Moreover, secretory rate was significantly higher at 10 4aM-melittin than at 5 ftM, and significantly higher at 5 /tM-melittin than at 2 ,UM. Similarly, as can be seen in Fig. 2, melittin stimulated PGE2 release in a concentration-dependent manner. Each point on this dose-response curve differs significantly from the control or basal release rate (142 + 8 ng g-' (60 min)-', shown as the filled bar, and release rate was significantly higher at 10 ,uM-melittin than at 5 /1M, and significantly higher at 5 /tM-melittin than at 2 aM. Collectively, the results in Figs 1 and 2 suggest the existence of a direct relationship between PGE2 release and renin secretory rate - as PGE2 release increases, renin secretion increases. Indeed, the dose-response curves are super-
P. C. CHURCHILL AND OTHERS
236 50 -
E
40-
0
CD
v 300 co
-W
4u 0 0
I
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._
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005
Melittin (#M)
Fig. 1. Melittin stimulates rat renal cortical slices to secrete renin in a concentrationdependent mainner. Each point on the dose-response curve represents the mean+S.E.M. of twelve to twenty-four experiments. Basal renin secretory rate+ one S.E.M. (n = 54) is represented by the height of the filled bar. Each point on the dose-response curve differs significantly from the basal secretory rate (P < 0-01 maximum). 2500c
.E
20001500-
-
CD 0
1000-
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ui
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O
i-
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Melittin (pM) Fig. 2. Melittin stimulates the release of PGE2 from rat renal cortical slices in a concentration-dependent manner. Each point on the dose-response curve represents the mean + S.E.M. of twelve to twenty-four experiments. Basal release + one S.E.M. (n = 72) is represented by the height of the filled bar. Each point on the dose-response curve differs significantly from the basal release rate (P < 0-0001 maximum).
imposable. However, there is no causal relationship between PGE2 release and renin secretory rate, as can be seen by comparing the results in Figs 3 and 4. Neither quinacrine nor meclofenamate had significant effects on basal or melittin-stimulated renin secretion, but both antagonized basal and melittin-stimulated PGE2 release. Both ouabain (Churchill, 1979) and TPA (Churchill & Churchill, 1984) have been
RENIN AND PROSTAGLANDIN E2 RELEASE No antagonist
Quinacrine
Meclofenamate
E
o 40co
D 30-
20-
0 C
pM-melittin in 5,uM-melittin Fig. 3. Renin secretory rates of rat renal cortical slices. Each bar represents the mean + S.E.M. of a given set of experiments, with n ranging from 12 to 54. The three bars grouped at the left show that 1 /SM-melittin (hatched bar) and 5 /LM-melittin (crosshatched bar) stimulate renin secretion in comparison with the basal rate (open bar). The bars grouped at the centre and at the right show that 20 /,M-quinacrine and 50 /immeclofenamate, respectively, had no significant effects on either basal or melittinstimulated renin secretion (P < 0-2 minimum compared with the corresponding values in the absence of quinacrine or meclofenamate). a No melittin
25001
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tm 1
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Meclofenamate
*c 2000 0
co 1500 0
co a 0)
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w
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0-cL No melittin en 1 lM-melittin S5M-melittin Each bar represents the renal cortical slices. rat 4. Rates of release from PGE2 Fig. mean + S.E.M. of a given set of experiments, with n ranging from 12 to 72. The three bars grouped at the left show that /LM-melittin (hatched bar) and 5 ,SM-melittin (crosshatched bar) stimulate PGE2 release in comparison with the basal release rate (open bar). The bars grouped at the centre and at the right show that 20 /SM-quinacrine and 50 zmmeclofenamate, respectively, inhibited both basal and melittin-stimulated PGE2 release (* P < 005 maximum compared with the corresponding values in the absence of quinacrine or meclofenamate).
237
P. C. CHLTR CHILL AND OTHERS
238
shown to inhibit basal renin secretory rate. These previous results were confirmed in the present studies (Fig. 5). However, whereas ouabain antagonized melittinstimulated renin secretion, TPA actually enhanced renin secretion in the presence of 5 /tm-melittin. Ouabain
No antagonist
TPA
5040-
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m
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s
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Fig. 5. Renin secretory rates of rat renal cortical slices. Each bar represents the mean + S.E.M. of a given set of experiments, with n ranging from 12 to 54. The three bars grouped at the left show that 1 uM-melittin (hatched bar) and 5 ,LM-melittin (crosshatched bar) stimulate renin secretion in comparison with the basal rate (open bar). The bars grouped at the centre show that 1 /tM-ouabain inhibited both basal and melittinstimulated renin secretion (*P < 0005 maximum compared with the corresponding values in the absence of ouabain). The bars grouped at the right show that 10 Jm,-12-0tetradecanoylphorbol 13-acetate (TPA) antagonized basal renin secretion but stimulated renin secretion in the presence of 5 /LM-melittin (* P < 0-05 maximum compared with the corresponding values in the absence of TPA). DISCUSSION
Melittin stimulated PGE2 synthesis by rat renal cortical slices in a concentrationdependent manner, and quinacrine antagonized basal as well as melittin-stimulated PGE2 synthesis. These results are in accord with expectations based on the observations that, in all cells studied so far, melittin stimulated phospholipase A2 (Hassid & Levine, 1977; Shier, 1979; Argiolas & Pisano, 1983), the first and centrally important step in the arachidonic acid cascade (Flower & Blackwell, 1976; Lapetina, 1982), and that quinacrine antagonizes phospholipase A2 activity (Flower & Blackwell, 1976; Craven, Briggs & DeRubertis, 1980; Wallach & Brown, 1981). The concentrations of melittin and quinacrine that produced effects on PGE2 synthesis in the present studies are in accord with these mechanisms of action. The threshold concentration for melittin-stimulation of phospholipase A2 activity, arachidonic acid release, and PG synthesis is approximately 1 /M (Hassid & Levine, 1977; Shier, 1979; Heisler et al. 1982; Argiolas & Pisano, 1983; Tanaka et al. 1983), and all of these effects are concentration-dependent. Similarly, 10-100 /tM concentrations of quina-
RENIN AND PROSTAGLANDIN E2 RELEASE
239
crine have concentration-dependent inhibitory effects on phospholipase A2 activity, arachidonic acid release. and PG synthesis (Flower & Blackwell, 1976; Craven et al. 1980; Wallach & Brown, 1981; Tanaka et al. 1983). Although melittin stimulated PGE2 synthesis and renin release simultaneously, and both effects were concentration dependent over the same range of concentrations, the results offer no support for the hypothesis that PG synthesis causes or mediates increased renin secretion. Both quinacrine and meclofenamate antagonized basal and melittin-simulated PGE2 synthesis, but neither quinacrine nor meclofenamate affected basal or melittin-stimulated renin release. Since quinacrine and meclofenamate antagonize PG synthesis by completely different mechanisms of action (inhibition of phospholipase A2 activity and decreased availability of substrate for PG synthesis versus inhibition of cyclo-oxygenase activity and decreased conversion of substrate to PGs), it is unlikely that the synthesis of any product of arachidonic acid is directly related to renin release. The mechanism of melittin-stimulated renin release, or secretion, is not clear. The previous results of others suggest at least three possibilities, which, however, seem to be contradicted by one or more of the present findings. First, melittin can produce such prolonged activation of phospholipase A2 that some cells have been observed to '... dissolve (themselves) in detergent lipids generated from (their) own membranes' (Shier, 1979). Thus, melittin-stimulated renin release could be viewed as increased leak of intracellular renin across damaged cell membranes. However, quinacrine inhibits phospholipase A2 activity. Therefore it should have blocked such a mechanism of action, but quinacrine failed to even antagonize melittin-stimulated renin release despite evidence that it was antagonizing phospholipase A2 activity (i.e. it decreased PGE2 release). Moreover, ouabain antagonized melittin-stimulated renin release, and if anything, it probably stimulates rather than inhibits phospholipase A2 activity (Churchill et al. 1985). Second, melittin has been shown to stimulate the release of renin bound to microsomal preparations of the renal cortex (Nishimura et al. 1980; Nishimura et al. 1980). However, activation of phospholipase A2 was implicated in producing this effect also. Therefore, this mechanism of action seems to be excluded by the results obtained with quinacrine, which suggest that melittin stimulates renin release or secretion independently of any action of melittin on phospholipase A2 activity. In addition, our results showed that melittin neither activates already secreted renin nor increases the rate of angiotensin I production from renin substrate. Third, mellitin inhibits protein kinase C activity (Nishizuka, 1984), and such an effect could conceivably stimulate renin secretion, since several stimulators of protein kinase C inhibit renin secretion (Churchill & Churchill, 1984; Kurtz et al. 1986). However, TPA is a potent stimulator of protein kinase C activity, and although it inhibited basal renin secretion in accord with previous findings (Churchill & Churchill, 1984). if anything, it enhanced renin secretion in the presence of 5 ftMmelittin. Interestingly, although the significance is unclear at the present time, melittinstimulated renin secretion differs in at least one respect from renin secretion stimulated by many other diverse substances. Ouabain antagonized but failed to completely block the effect of melittin, but ouabain has been shown to reduce the
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renin secretory rate in this preparation to virtually zero, despite the presence of a variety of secretory stimulants including ,-adrenergic agonists, A2 adenosine receptor agonists, phosphodiesterase inhibitors, dibutyryl cyclic AMP, phenytoin. and combinations of these (reviewed by Churchill, 1985). In conclusion, melittin stimulates PGE2 synthesis and renin secretion of rat renal cortical slices, but these effects are not causally related. The effects of PGE2 synthesis, but not the effects on renin secretion, appear to be mediated by activation of phospholipase A2. The mechanism by which melittin stimulates renin secretion remains to be determined, but whatever the mechanism, it cannot be completely blocked by ouabain. This research was supported by the National Institutes of Health (HL 24880). We thank Parke Davis-Warner Lambert Co. (Ann Arbor. MI, USA) for the generous supply of meclofenamate. REFERENCES
ARGIOLAS, A. & PISANO, J. J. (1983). Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom. Journal of Biological Chemistry 258, 13697-13702.
BEIERWALTES, W. H., SCHRYVER, S., OLSON, P. S. & ROMERO, J. C. (1980). Interaction of the prostaglandin and renin-angiotensin systems in isolated rat glomeruli. American Journal of Physiology 239, F602-608. BEIERWALTES7 W. H., SCHRYVER, S., SANDERS, E., STRAND, J. & ROMERO. J. C. (1982). Renin release selectively stimulated by prostaglandin-12 in isolated rat glomeruli. American Journal of Physiology 243, F276-283. CHURCHILL. P. C. (1979). Possible mechanism of the inhibitory effect of ouabain on renin secretion from rat renal cortical slices. Journal of Physiology 294. 123-134. CHURCHILL, P. C. (1985). Second messengers in renin secretion. American Journal of Physiology 249, F 175-184. CHURCHILL, P. C. & CHURCHILL, M. C. (1984). 12-O-Tetradecanoylphorbol 13-acetate inhibits renin secretion of rat renal cortical slices. Journal of Hypertension 2, suppl. 1. 25-28. CHURCHILL, P. C., CHURCHILL, M. C. & MCDONALD, F. D. (1985). Quinacrine antagonizes the effects of Na,K-ATPase inhibitors on renal prostaglandin E2 release but not their effects on renin secretion. Life Sciences 36, 277-282. CHURCHILL, P. C., SAVOY-MOORE, R. T. & CHURCHILL, M. C. (1983). Lack of relationship between prostaglandin E2 release and renin secretion in rat renal cortical slices. Journal of Pharmacology and Experimental Therapeutics 226, 46-51. CRAVEN, P. A., BRIGGS, R. & DERUBERTIS, F. R. (1980). Calcium-dependent action of osmolalitv on adenosine 3'.5'-monophosphate accumulation in rat renal inner medulla. Jouirnal of Clinical Investigation 65. 529-542. FLOWER, R. J. & BLACKWELL, G. J. (1976). The importance of phospholipase-A2 in prostaglandin biosynthesis. Biochemical Pharmacology 25, 285-291. FREEMAN, R. H., DAVIS, J. 0. & VILLARREAL, D. (1984). Role of renal prostaglandins in the control of renin release. Circulation Research 54, 1-9. HASSID, A. & LEVINE. L. (1977). Stimulation of phospholipase activitv and prostaglandin biosynthesis by melittin in cell culture and in vivo. Research Communications in Chemistry, Pathology and Pharmacology 18, 507-517. HEISLER, S., REISINE. T. D., HOOK, V. Y. H. & AXELROD, J. (1982). Somatostatin inhibits multireceptor stimulation of cyclic AMP formation and corticotropin secretion in mouse pituitary tumor cells. Proceedings of the NVational Academy of Sciences of the USA 79, 6502-6506. HENRICH, W. L. (1981). Role of prostaglandins in renin secretion. Kidney International 19, 822-830. KEETON, T. K. & CAMPBELL, W. B. (1980). The pharmacologic alteration of renin release. Pharmacological Review 32, 81-227.
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KURTZ, A., PFEILSCHIFTER, J.. HUTTER. A., BUHRLE, C., NOBILING, R., TAUGNER. R., HACKENTHAL, E. & BAUER, C. (1986). Role of protein kinase (C in inhibition of renin release caused by vasoconstrictors. American Journal of Physiology 250, C563-57 1. LAPETINA. E. G. (1982). Regulation of arachidonic acid production: role of phospholipases C and A2. Trends in Pharmacological Sciences 3, 115-118. NISHIMURA. K.. ALHENC-GELAS. F.. WHITE. A. & ERDOS, E. G. (1980). Activation of membranebound kallikrein and renin in the kidney. Proceedings of the National Academy of Sciences of the UtSA 77, 4975-4978. NISHIMURA. K.. WARD. P. & ERDOS, E. G. (1980). Kallikrein and renin in the membrane fractionls of the rat kidney. Hypertension 2, 538-545. NISHIZUKA, Y. (1984). Turnover of inositol phospholipids and signal transduction. Science 225, 1365-1369. SHIER. WV. T. (1979). Activation of high levels of endogenous phospholipase A2 in cultured cells. Proceedings of the National Academy of Sciences of the USA 76. 195-199. SUN, F. F.. TAYLOR. B. NM.. MNCGUIRE. J. C. & WONG. P. Y. K. (1981). Metabolism of prostaglaindins in the kidney. Kidney International 19. 760-770. TANAKA. N., KAGAWA, S., MURAKOSO, K.. SHIMIZU, S. & MATSUOKA, A. (1983). Enhancement of insulin release due to inhibition of phospholipase A2 activity. Hormone and Metabolic Research 15, 255-256. WALLACH. D. P. & BROWN. V. J. R. (1981). Studies on the arachidonic acid cascade. I. Inhibition of phospholipase A2 in vitro and in vivo by several novel series of inhibitor compounds. Biochemical Pharmacology 30, 1315-1324. WVALLENSTEIN, S., ZUCKER, C. L. & FLEISS, J. L. (1980). Some statistical methods useful in circulation research. Circulation Research 47. 1-9.
Journal of Physiology (1990). 428. pp. 233-241 W'ith 5 fig res Prinited in Great Britain
EFFECT OF MELITTIN ON RENIN AND PROSTAGLANDIN E2 RELEASE FROM RAT RENAL CORTICAL SLICES
BY PAUL C. CHURCHILL, NOREEN F. ROSSI, MONIQUE C. CHURCHILL AND VIRGINIA R. ELLIS From the Department of Physiology and Internal Medicine, Wayne State University School of Medicine, Detroit, MIU 48201, UTSA
(Received 16 February 1990) SUMMARY
1. The present experiments were designed to determine the effect of melittin on renin secretion. Melittin is a polypeptide component of bee venom which stimulates phospholipase A2 activity, thereby increasing arachidonic acid release and prostaglandin (PG) synthesis, and which inhibits protein kinase C activity. Either of these actions might be expected to stimulate renin secretion, since renin secretion is stimulated by arachidonic acid and by several PGs, and since renin secretion is inhibited by several activators of protein kinase C. 2. In rat renin cortical slices incubated at 37 'C in a buffered and oxygenated physiological saline solution, 0 -0l uM-melittin produced a concentration-dependent stimulation of both prostaglandin E2 (PGE2) synthesis and renin secretion. However, melittin-stimulated renin secretion is independent of melittin-stimulated phospholipase A2 activity, arachidonic acid release, and PG synthesis, since 20 ,umquinacrine (a phospholipase A2 antagonist) and 50 /tM-meclofenamate (a cyclooxygenase antagonist) antagonized basal and melittin-stimulated PGE2 synthesis but had no effects on basal or melittin-stimulated renin secretion. 3. Furthermore, melittin-stimulated renin secretion is not produced by inhibition of protein kinase C, since an activator of protein kinase C (12-O-tetradecanoylphorbol 13-acetate, TPA), enhanced rather than antagonized melittin-stimulated renin secretion. Ouabain partially antagonized, but did not completely block, melittinstimulated renin secretion. 4. Thus, melittin-stimulated phospholipase A2 activity probably accounts for stimulated PGE2 production, but not for stimulated renin secretion. The mechanism of melittin-stimulated renin secretion is unclear; an effect on protein kinase C does not appear to be involved, and in contrast to the stimulatory effects of a variety of other substances, melittin-stimulated renin secretion is only partially antagonized by ouabain. INTRODUCTION
Both the renal cortex and the renal medulla synthesize PGs (Sun, Taylor, McGuire & Wong, 1981), and several investigators have suggested that endogenous PGs play a role in the physiological control of renin secretion (Beierwaltes, Schryver, .NIS 8281
234
P. C CHURCHILL AND OTHERS
Olson & Romero, 1980; Keeton & Campbell, 1980; Henrich, 1981; Beierwaltes,
Schryver, Sanders, Strand & Romero, 1982; Freeman, Davis & Villarreal. 1984). Three lines of evidence support this suggestion (reviewed by Henrich, 1981). First, many exogenous (PGs have been shown to stimulate renin secretion in vivo, and in a wide variety of in vitro preparations including isolated glomeruli (Beierwaltes et al. 1982). Effects in the latter preparation demonstrate that the effects are exerted directly, independently of PG-induced changes in renal haemodynamics and/or the renal tubular handling of NaCl. Second, exogenous arachidonic acid stimulates renin secretion in vivo and Hin vitro, and this response is mediated by newly synthesized PGs. since it is antagonized by cyclo-oxygenase inhibitors. Third, cyclo-oxygenase inhibitors have been shown to suppress both basal and stimulated renin secretion in vivo. Collectively, these observations have led some investigators to hypothesize that PG synthesis mediates renin secretion (Henrich, 1981). The present experiments were designed to further explore the relationship between renin secretion and PG synthesis using melittin as a probe. Melittin is a polypeptide component of bee venom which, in all cells studied to date, activates phospholipase A2 activity, thereby increasing arachidonic acid release and PG synthesis (Hassid & Levine, 1977; Argiolas & Pisano, 1983; Tanaka, Kagawa, Murakoso, Shimizu & Matsuoka, 1983). Although there are no previous reports of its effects on renin secretion per se, melittin has been shown to release membrane-bound renin (Nishimura, Alhenc-Gelas, White & Erdos, 1980; Nishimura, XWard & Erdos, 1980), and to stimulate the release of other stored secretory products (Heisler, Reisine, Hook & Axelrod, 1982; Tanaka et al. 1983), observations which suggest that it might stimulate both renin secretion and PG synthesis. Also, melittin might be expected to stimulate renin secretion by another mechanism, independently of phospholipase A2 activity, arachidonic acid release, and PG synthesis: melittin inhibits protein kinase C activity (Nishizuka, 1984), and several observations suggest an inverse relationship between renin secretion and protein kinase C activity (Churchill & Churchill, 1984; Kurtz, Pfeilschifter, Hutter, Buhrle, Nobiling, Taugner, Hackenthal & Bauer, 1986). Therefore, experiments were designed to determine the effect, and the mechanism of action, of melittin on renin secretion. METHODS
The procedures for studying renin and PGE2 release using rat renal cortical slices have been described in detail previously (Churchill. 1979; Churchill, Savoy-MNoore & Churchill. 1983). For each of several experiments, five ether-anaesthetized adult male Sprague-Dawley rats acted as kidney donors. After bilateral nephrectomy, the rats were killed with ether and bilateral pneumothorax. The renal capsule was removed from each kidney. and using a razor blade, four thin slices were cut. The resulting forty slices were distributed between incubation flasks (two slices per flask) each of which contained 12 ml of incubation medium at 37 °C, previously equilibrated with a mixture of 02 (95%) and CO2 (5%). The flasks were stoppered and placed in a oscillating water bath maintained at 37 'C. The gas phase in each flask was continuously replaced throughout the incubation period. Samples of media (1 ml) were taken at 30. 60 and 90 min. The samples were centrifuged (4 'C; 8000 g; 5 min) and the supernatants were stored at -70 'C until analysed for renin and PGE2. After the incubations. tissue dry weight was determined. Dry weights ranged between 0-58 and 6-40 mg per flask (two slices) and averaged 2-95+0-08 mg (n = 318 flasks). The composition of the control incubation medium was: NaCl, 125 mM; NaHCO3 19 mm; KCl, 4 mM; CaCl2, 2-5 mm; NaH2PO4, 1-2 mm; MgSO4. 08 mM; and 0-2 g (100 ml)-' each of glucose and bovine serum albumin. Dose-response curves were generated using melittin concentrations ranging
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from 01 to 10 /IM. Meclofenamate, ouabain (Strophanthin G), quinacrine, and 12-0-tetradecanoylphorbol 13-acetate (TPA) were used in some experiments at concentrations which were shown in previous experiments to produce maximal effects (Churchill et al. 1983; Churchill & Churchill, 1984; Churchill, Churchill & McDonald, 1985). A set of control slices was run simultaneously with experimental slices. Renin concentration of the incubation medium was determined using a modification (Churchill et al. 1983) of a previously described method (Churchill, 1979). In brief, renin standards and renincontaining supernatants were incubated with rat renin substrate at 37 °C for 30 min. The angiotensin I generated during this incubation was determined by radioimmunoassay. Renin concentration was calculated as nanograms angiotensin I generated per millilitre of renincontaining sample per hour of incubation with renin substrate (ng ml-' h-'). There was a linear relationship between the renin concentration of the standards expressed as ng ml-' h-' and expressed as Goldblatt Units per millilitre (GU ml-'). This relationship was used to convert renin concentrations of the unknowns from ng ml-' h-' to GU ml-'. The total renin secreted at a given time during the incubation of the slices was calculated as the renin concentration of the medium (GU ml-') multiplied by the volume of the medium at the time of sampling (e.g. 12 ml at 30 min, etc.) and divided by tissue dry weight, yielding the units GU g-'. Renin secretory rate was calculated as the increment in the total amount of renin during the final hour of incubation (from 30 to 90 min), and expressed as GU g-' (60 min)-'. The first 30 min period was taken as an
equilibration period. A melittin-free sample of renin-containing incubation medium was divided into two equal portions. Melittin was added to one portion (1O IM final concentration), and both portions were placed in the 37 °C water bath for 90 min to simulate the conditions for incubating kidney slices. Then, six aliquots of each portion were incubated with renin substrate as described above, and angiotensin I in each of these was determined in duplicate by radioimmunoassay. Renin concentrations of melittin-free and melittin-containing samples averaged 20-4 ±1-4 and 16-9 + 1-5 ng ml-' h-'. respectively. These means do not differ significantly (P > 0 1). Therefore, melittin neither activates an inactive form of renin nor affects the reaction between renin and renin substrate. PGE2 in supernatants was measured using a commercial radioimmunoassay kit (New England Nuclear) without any preprocessing, as discussed previously (Churchill et al. 1983). The total amount released by the slices was calculated as the concentration in the medium (ng ml-'), multiplied by the volume of the medium at the time of sampling (e.g. 12 ml at 30 min, etc.), and divided by tissue dry weight (g) yielding the units ng g-'. Release rate was calculated as the increment in ng g-' during the final hour of incubation, and expressed in the units ng g-' (60 min)-'. All results are expressed as means + S.E.M.s. Analysis of variance and Scheffe contrasts (Wallenstein, Zucker & Fleiss, 1980) were used to assess the statistical significance of differences between means. P values < 0-05 were considered statistically significant. RESULTS
Control or basal renin secretory rate averaged 106 +0-4 GU g-1 (60 min)-' (n = 54). As can be seen in Fig. 1, melittin stimulated renin secretory rate in a concentration-dependent manner. Each point on the dose-response curve differs significantly from the control or basal secretory rate, shown as the filled bar. Moreover, secretory rate was significantly higher at 10 4aM-melittin than at 5 ftM, and significantly higher at 5 /tM-melittin than at 2 ,UM. Similarly, as can be seen in Fig. 2, melittin stimulated PGE2 release in a concentration-dependent manner. Each point on this dose-response curve differs significantly from the control or basal release rate (142 + 8 ng g-' (60 min)-', shown as the filled bar, and release rate was significantly higher at 10 ,uM-melittin than at 5 /1M, and significantly higher at 5 /tM-melittin than at 2 aM. Collectively, the results in Figs 1 and 2 suggest the existence of a direct relationship between PGE2 release and renin secretory rate - as PGE2 release increases, renin secretion increases. Indeed, the dose-response curves are super-
P. C. CHURCHILL AND OTHERS
236 50 -
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-W
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._
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Fig. 1. Melittin stimulates rat renal cortical slices to secrete renin in a concentrationdependent mainner. Each point on the dose-response curve represents the mean+S.E.M. of twelve to twenty-four experiments. Basal renin secretory rate+ one S.E.M. (n = 54) is represented by the height of the filled bar. Each point on the dose-response curve differs significantly from the basal secretory rate (P < 0-01 maximum). 2500c
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Melittin (pM) Fig. 2. Melittin stimulates the release of PGE2 from rat renal cortical slices in a concentration-dependent manner. Each point on the dose-response curve represents the mean + S.E.M. of twelve to twenty-four experiments. Basal release + one S.E.M. (n = 72) is represented by the height of the filled bar. Each point on the dose-response curve differs significantly from the basal release rate (P < 0-0001 maximum).
imposable. However, there is no causal relationship between PGE2 release and renin secretory rate, as can be seen by comparing the results in Figs 3 and 4. Neither quinacrine nor meclofenamate had significant effects on basal or melittin-stimulated renin secretion, but both antagonized basal and melittin-stimulated PGE2 release. Both ouabain (Churchill, 1979) and TPA (Churchill & Churchill, 1984) have been
RENIN AND PROSTAGLANDIN E2 RELEASE No antagonist
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o 40co
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pM-melittin in 5,uM-melittin Fig. 3. Renin secretory rates of rat renal cortical slices. Each bar represents the mean + S.E.M. of a given set of experiments, with n ranging from 12 to 54. The three bars grouped at the left show that 1 /SM-melittin (hatched bar) and 5 /LM-melittin (crosshatched bar) stimulate renin secretion in comparison with the basal rate (open bar). The bars grouped at the centre and at the right show that 20 /,M-quinacrine and 50 /immeclofenamate, respectively, had no significant effects on either basal or melittinstimulated renin secretion (P < 0-2 minimum compared with the corresponding values in the absence of quinacrine or meclofenamate). a No melittin
25001
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co a 0)
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0-cL No melittin en 1 lM-melittin S5M-melittin Each bar represents the renal cortical slices. rat 4. Rates of release from PGE2 Fig. mean + S.E.M. of a given set of experiments, with n ranging from 12 to 72. The three bars grouped at the left show that /LM-melittin (hatched bar) and 5 ,SM-melittin (crosshatched bar) stimulate PGE2 release in comparison with the basal release rate (open bar). The bars grouped at the centre and at the right show that 20 /SM-quinacrine and 50 zmmeclofenamate, respectively, inhibited both basal and melittin-stimulated PGE2 release (* P < 005 maximum compared with the corresponding values in the absence of quinacrine or meclofenamate).
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shown to inhibit basal renin secretory rate. These previous results were confirmed in the present studies (Fig. 5). However, whereas ouabain antagonized melittinstimulated renin secretion, TPA actually enhanced renin secretion in the presence of 5 /tm-melittin. Ouabain
No antagonist
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Fig. 5. Renin secretory rates of rat renal cortical slices. Each bar represents the mean + S.E.M. of a given set of experiments, with n ranging from 12 to 54. The three bars grouped at the left show that 1 uM-melittin (hatched bar) and 5 ,LM-melittin (crosshatched bar) stimulate renin secretion in comparison with the basal rate (open bar). The bars grouped at the centre show that 1 /tM-ouabain inhibited both basal and melittinstimulated renin secretion (*P < 0005 maximum compared with the corresponding values in the absence of ouabain). The bars grouped at the right show that 10 Jm,-12-0tetradecanoylphorbol 13-acetate (TPA) antagonized basal renin secretion but stimulated renin secretion in the presence of 5 /LM-melittin (* P < 0-05 maximum compared with the corresponding values in the absence of TPA). DISCUSSION
Melittin stimulated PGE2 synthesis by rat renal cortical slices in a concentrationdependent manner, and quinacrine antagonized basal as well as melittin-stimulated PGE2 synthesis. These results are in accord with expectations based on the observations that, in all cells studied so far, melittin stimulated phospholipase A2 (Hassid & Levine, 1977; Shier, 1979; Argiolas & Pisano, 1983), the first and centrally important step in the arachidonic acid cascade (Flower & Blackwell, 1976; Lapetina, 1982), and that quinacrine antagonizes phospholipase A2 activity (Flower & Blackwell, 1976; Craven, Briggs & DeRubertis, 1980; Wallach & Brown, 1981). The concentrations of melittin and quinacrine that produced effects on PGE2 synthesis in the present studies are in accord with these mechanisms of action. The threshold concentration for melittin-stimulation of phospholipase A2 activity, arachidonic acid release, and PG synthesis is approximately 1 /M (Hassid & Levine, 1977; Shier, 1979; Heisler et al. 1982; Argiolas & Pisano, 1983; Tanaka et al. 1983), and all of these effects are concentration-dependent. Similarly, 10-100 /tM concentrations of quina-
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crine have concentration-dependent inhibitory effects on phospholipase A2 activity, arachidonic acid release. and PG synthesis (Flower & Blackwell, 1976; Craven et al. 1980; Wallach & Brown, 1981; Tanaka et al. 1983). Although melittin stimulated PGE2 synthesis and renin release simultaneously, and both effects were concentration dependent over the same range of concentrations, the results offer no support for the hypothesis that PG synthesis causes or mediates increased renin secretion. Both quinacrine and meclofenamate antagonized basal and melittin-simulated PGE2 synthesis, but neither quinacrine nor meclofenamate affected basal or melittin-stimulated renin release. Since quinacrine and meclofenamate antagonize PG synthesis by completely different mechanisms of action (inhibition of phospholipase A2 activity and decreased availability of substrate for PG synthesis versus inhibition of cyclo-oxygenase activity and decreased conversion of substrate to PGs), it is unlikely that the synthesis of any product of arachidonic acid is directly related to renin release. The mechanism of melittin-stimulated renin release, or secretion, is not clear. The previous results of others suggest at least three possibilities, which, however, seem to be contradicted by one or more of the present findings. First, melittin can produce such prolonged activation of phospholipase A2 that some cells have been observed to '... dissolve (themselves) in detergent lipids generated from (their) own membranes' (Shier, 1979). Thus, melittin-stimulated renin release could be viewed as increased leak of intracellular renin across damaged cell membranes. However, quinacrine inhibits phospholipase A2 activity. Therefore it should have blocked such a mechanism of action, but quinacrine failed to even antagonize melittin-stimulated renin release despite evidence that it was antagonizing phospholipase A2 activity (i.e. it decreased PGE2 release). Moreover, ouabain antagonized melittin-stimulated renin release, and if anything, it probably stimulates rather than inhibits phospholipase A2 activity (Churchill et al. 1985). Second, melittin has been shown to stimulate the release of renin bound to microsomal preparations of the renal cortex (Nishimura et al. 1980; Nishimura et al. 1980). However, activation of phospholipase A2 was implicated in producing this effect also. Therefore, this mechanism of action seems to be excluded by the results obtained with quinacrine, which suggest that melittin stimulates renin release or secretion independently of any action of melittin on phospholipase A2 activity. In addition, our results showed that melittin neither activates already secreted renin nor increases the rate of angiotensin I production from renin substrate. Third, mellitin inhibits protein kinase C activity (Nishizuka, 1984), and such an effect could conceivably stimulate renin secretion, since several stimulators of protein kinase C inhibit renin secretion (Churchill & Churchill, 1984; Kurtz et al. 1986). However, TPA is a potent stimulator of protein kinase C activity, and although it inhibited basal renin secretion in accord with previous findings (Churchill & Churchill, 1984). if anything, it enhanced renin secretion in the presence of 5 ftMmelittin. Interestingly, although the significance is unclear at the present time, melittinstimulated renin secretion differs in at least one respect from renin secretion stimulated by many other diverse substances. Ouabain antagonized but failed to completely block the effect of melittin, but ouabain has been shown to reduce the
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renin secretory rate in this preparation to virtually zero, despite the presence of a variety of secretory stimulants including ,-adrenergic agonists, A2 adenosine receptor agonists, phosphodiesterase inhibitors, dibutyryl cyclic AMP, phenytoin. and combinations of these (reviewed by Churchill, 1985). In conclusion, melittin stimulates PGE2 synthesis and renin secretion of rat renal cortical slices, but these effects are not causally related. The effects of PGE2 synthesis, but not the effects on renin secretion, appear to be mediated by activation of phospholipase A2. The mechanism by which melittin stimulates renin secretion remains to be determined, but whatever the mechanism, it cannot be completely blocked by ouabain. This research was supported by the National Institutes of Health (HL 24880). We thank Parke Davis-Warner Lambert Co. (Ann Arbor. MI, USA) for the generous supply of meclofenamate. REFERENCES
ARGIOLAS, A. & PISANO, J. J. (1983). Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom. Journal of Biological Chemistry 258, 13697-13702.
BEIERWALTES, W. H., SCHRYVER, S., OLSON, P. S. & ROMERO, J. C. (1980). Interaction of the prostaglandin and renin-angiotensin systems in isolated rat glomeruli. American Journal of Physiology 239, F602-608. BEIERWALTES7 W. H., SCHRYVER, S., SANDERS, E., STRAND, J. & ROMERO. J. C. (1982). Renin release selectively stimulated by prostaglandin-12 in isolated rat glomeruli. American Journal of Physiology 243, F276-283. CHURCHILL. P. C. (1979). Possible mechanism of the inhibitory effect of ouabain on renin secretion from rat renal cortical slices. Journal of Physiology 294. 123-134. CHURCHILL, P. C. (1985). Second messengers in renin secretion. American Journal of Physiology 249, F 175-184. CHURCHILL, P. C. & CHURCHILL, M. C. (1984). 12-O-Tetradecanoylphorbol 13-acetate inhibits renin secretion of rat renal cortical slices. Journal of Hypertension 2, suppl. 1. 25-28. CHURCHILL, P. C., CHURCHILL, M. C. & MCDONALD, F. D. (1985). Quinacrine antagonizes the effects of Na,K-ATPase inhibitors on renal prostaglandin E2 release but not their effects on renin secretion. Life Sciences 36, 277-282. CHURCHILL, P. C., SAVOY-MOORE, R. T. & CHURCHILL, M. C. (1983). Lack of relationship between prostaglandin E2 release and renin secretion in rat renal cortical slices. Journal of Pharmacology and Experimental Therapeutics 226, 46-51. CRAVEN, P. A., BRIGGS, R. & DERUBERTIS, F. R. (1980). Calcium-dependent action of osmolalitv on adenosine 3'.5'-monophosphate accumulation in rat renal inner medulla. Jouirnal of Clinical Investigation 65. 529-542. FLOWER, R. J. & BLACKWELL, G. J. (1976). The importance of phospholipase-A2 in prostaglandin biosynthesis. Biochemical Pharmacology 25, 285-291. FREEMAN, R. H., DAVIS, J. 0. & VILLARREAL, D. (1984). Role of renal prostaglandins in the control of renin release. Circulation Research 54, 1-9. HASSID, A. & LEVINE. L. (1977). Stimulation of phospholipase activitv and prostaglandin biosynthesis by melittin in cell culture and in vivo. Research Communications in Chemistry, Pathology and Pharmacology 18, 507-517. HEISLER, S., REISINE. T. D., HOOK, V. Y. H. & AXELROD, J. (1982). Somatostatin inhibits multireceptor stimulation of cyclic AMP formation and corticotropin secretion in mouse pituitary tumor cells. Proceedings of the NVational Academy of Sciences of the USA 79, 6502-6506. HENRICH, W. L. (1981). Role of prostaglandins in renin secretion. Kidney International 19, 822-830. KEETON, T. K. & CAMPBELL, W. B. (1980). The pharmacologic alteration of renin release. Pharmacological Review 32, 81-227.
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KURTZ, A., PFEILSCHIFTER, J.. HUTTER. A., BUHRLE, C., NOBILING, R., TAUGNER. R., HACKENTHAL, E. & BAUER, C. (1986). Role of protein kinase (C in inhibition of renin release caused by vasoconstrictors. American Journal of Physiology 250, C563-57 1. LAPETINA. E. G. (1982). Regulation of arachidonic acid production: role of phospholipases C and A2. Trends in Pharmacological Sciences 3, 115-118. NISHIMURA. K.. ALHENC-GELAS. F.. WHITE. A. & ERDOS, E. G. (1980). Activation of membranebound kallikrein and renin in the kidney. Proceedings of the National Academy of Sciences of the UtSA 77, 4975-4978. NISHIMURA. K.. WARD. P. & ERDOS, E. G. (1980). Kallikrein and renin in the membrane fractionls of the rat kidney. Hypertension 2, 538-545. NISHIZUKA, Y. (1984). Turnover of inositol phospholipids and signal transduction. Science 225, 1365-1369. SHIER. WV. T. (1979). Activation of high levels of endogenous phospholipase A2 in cultured cells. Proceedings of the National Academy of Sciences of the USA 76. 195-199. SUN, F. F.. TAYLOR. B. NM.. MNCGUIRE. J. C. & WONG. P. Y. K. (1981). Metabolism of prostaglaindins in the kidney. Kidney International 19. 760-770. TANAKA. N., KAGAWA, S., MURAKOSO, K.. SHIMIZU, S. & MATSUOKA, A. (1983). Enhancement of insulin release due to inhibition of phospholipase A2 activity. Hormone and Metabolic Research 15, 255-256. WALLACH. D. P. & BROWN. V. J. R. (1981). Studies on the arachidonic acid cascade. I. Inhibition of phospholipase A2 in vitro and in vivo by several novel series of inhibitor compounds. Biochemical Pharmacology 30, 1315-1324. WVALLENSTEIN, S., ZUCKER, C. L. & FLEISS, J. L. (1980). Some statistical methods useful in circulation research. Circulation Research 47. 1-9.
