Regulation of inducible nitric oxide production by intracellular calcium

Regulation of inducible nitric oxide production by intracellular calcium Mark L. Jordan, MD, Barbara Rominski, BS, Andrea Jaquins-Gerstl, BS, David Geller, MD, and Rosemary A. Hoffman, BS, Pittsburgh, Pa.

Background. Because increased nitric oxide (NO) production during sepsis can be detrimental to the host, inhibition of NO synthesis by various antagonists has been proposed as a therapeutic option. However, none of these approaches has addressed the possible regulation of inducible NO synthesis (iNOS) by endogenous intraceUular signaling mechanisms. The purpose of our study was to determine whether intracellular calcium ([Ca2+]i) regulates iNOS. Methods. The macrophage cell line R A W 264.7 cells stimulated to produce NO were cultured in the presence or absence of the calcium ionophore A23187, ionomycin, or the Ca2§ triphosphatase inhibitor thapsigargin. Supernatants and total cellular RNA were recovered for measurement of iNOS messenger RNA (Northern blot.) and NO2- (stable end product of NO), respectively. Simultaneous measurement of [Ca2+]i was performed by fluorescence spectrophotometry. Results. The calcium ionophore A23187, ionomycin, and thapsigargin all produced a dose-dependent inhibition of NO end product and iNOS messenger RNA (confirmed by densitometry) with a simultaneous increase in [Ca2+]i, confirmed by fluorescence spectrophotometry. This could not be reversed by exogenous L-arginine and was not observed if these agents were added beyond 0 hours of culture. Conclusions. Although iNOS is traditionally viewed as calcium independent, these data clearly show that [Ca2+]i regulates iNOS messenger RNA induction and end product synthesis. Endogenous cellular second messenger may thus be important in autoregulation of NO synthesis. Alternatives to pharmacologic or antagonist inhibition of NO deserve further investigation. (SURaF_~Y 1995;118:138-46.) From the Meegan Lynch Hickey Lab for Life, Division of Urologic Surgery~Renal Transplantation, Department of Surgery, University of Pittsburgh Medical Center, and the Veterans Administration Medical Center, Pittsburgh, Pa.

NrrRIc OXIDE (NO) is thought to mediate several of the physiologic derangements observed in critically ill patients, resulting in changes in hemodynamic and metabolic function that may be detrimental to survival. 1-~As a result inhibition of NO synthesis has been suggested as a therapeutic option during sepsis and endotoxic shock.4, 5 In contrast, other studies have suggested that NO may provide beneficial effects during shock, including preservation of renal 6 and hepatic perfusion, 7 improvement in sepsis-induced pulmonary dysfunction, 8 and reduced lethality in endotoxemic animals. 9' 10 NO also exerts immunoregulatory effects that may have important implications for organ transplantation. 11, IS

MATERIAL A N D M E T H O D S

Supported by a Veterans AdministrationMerit Reviewgrant, the Meegan Lynch HickeyLab for Life, and the National Institutes of Health (2R37AI-16869-11). Presented at the Fifty-sixthAnnual Meeting of the Societyof University Surgeons, Denver, Colo., Feb. 9-11, 1995. Reprint requests: Mark L. Jordan, MD, Universityof Pittsburgh Medical Center, Suite 700, 3471 Fifth Ave., Pittsburgh, PA 15213. 11/6/64899

Cells. The macrophage cell line RAW 264.7 was grown to subconfluence in 175 cm z flasks and harvested by gentle scraping to remove adherent cells as previously described. 12 For some experiments 0.1 x 106 ceils were plated in medium (Dulbecco's modified Eagle's medium (DMEM) containing 0.6 m m o l / L L-arginine in triplicate 96-well microtiter plates for various periods in the presence or absence of interferon-~/ (IFN-~) 100 units/ml + lipopolysaccharide (LPS) 10 p g / m l (condi-

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The effects of N O in these systems are obviously complex, and the basic mechanisms regulating NO production are as yet incompletely understood. Because of our previous observations of the regulation of T-cell intracellular calcium ([Ca2+]i) by NO, 13 we hypothesized that regulation of inducible NO synthesis (iNOS) may occur at the level of intracellular [Ca2+]i signaling. Despite the traditional concept that iNOS is Ca ~+ independent, our results show that both NO end-product synthesis and messenger RNA (mRNA) induction are inhibited by agents that cause an increase in [Ca2+]i.

Surgery Volume 118, Number 2 tioned medium, CM) with or without test substances. For other experiments requiring harvesting of RNA, 20 x 106 cells were plated in duplicate 100 mm x 15 mm Petri dishes and cultured in the presence or absence of CM with or without test substances. Supernatant NO2- analysis. NO2 levels in culture supernamnts were analyzed with a microplate reader by using the Griess reaction 14with NaNO2- as the standard. [Ca2+]i measurement. Incubation of 1 • 107 RAW cells with the acetoxymethylester derivative of indo-1 (indo-1, 2 lamol/L; Molecular Probes, Eugene, Ore.) was carried out for 30 minutes at 37 ~ C in DMEM with 0.6 m m o l / L L-arginine in 10% CO2. Cells were washed and kept at I • 106/ml in DMEM at 37 ~ C. Immediately before use cells were washed in buffer (140 m m o l / L NaC1, 3 m m o l / L KC1, 1.8 m m o l / L CaC12, 1 m m o l / L MgC12, 10 m m o l / L glucose, and 25 m m o l / L HEPES; pH 7.23). The fluorescence emission of 2 x 106 cells was measured in 1.7 ml buffer in a stirred heated curvette in a Shimadzu RF5000U spectrofluorophotometer (Shimadzu Scientific Instruments, Columbia, Md.) with excitation at 331 nm and emission at 410 nm as previously described.15 Each run was calibrated by addition of0.1% Triton-X followed by egtazic acid (4 mmol/L) and Tris Base (40 mrnol/L). [Ca2+]i was calculated by the formula [Ca2+]i = K
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tions in amount of total RNA per lane. Relative mRNA levels were quantitated by a Betascope radioanalytic scanner (Betagen, Waltham, Mass.). Northern blot shown is representative of three separate experiments performed at different times. Statistical analysis. All data are expressed as the mean -+- SEM. Statistical comparisons were made with Student's t tests or analysis of variance where appropriate. All experiments were performed at least three times.

RESULTS Effects of calcium ionophore (CaI) on NO production. Supernatants from cultures of RAW cells were assayed for NO production as NO2- accumulation as previously described. 14 Preliminary experiments showed that maximal NO production occurred in the presence of 100 units/ml IFN-7 + 10 ttg/ml LPS (CM); hence these concentrations were used i n all subsequent experiments. Our previous work with RAW cells has also shown that mRNA for iNOS is fully induced in 6 hours and end product NO is optimal at 24 hours~8; hence these time points were used to study the effects of the calcium-active agents described in this and subsequent experiments. Supernatants from control RAW cell cultures conmining Cai (1 to 10 l~mol/L) or vehicle (0.5% dimethyl sulfoxide) corresponding to the highest concentration (10 pmol/L) of CaI tested alone without CM contained only low basal levels of NO2- (<3 ttmol/L) at all time points (data not shown). As shown in Fig. 1, CaI (0.3 to 10 pmol/L) added at the initiation of the culture period caused a dose-dependent decrease in NO2- accumulation at both 6 and 24 hours. NG-monomethyl-arginine (NMA), a competitive inhibitor of L-arginine metabolism, also significantly reduced NO production. Cell viability in the presence of either vehicle or CaI was confirmed by lactate dehydrogenase levels assayed in the same cells under the same conditions (not shown). Preincubation of the cells with CaI (2 Ilmol/L) for I hour followed by washing and culturing for 24 hours in the presence or absence of CM either with or without the same concentration of CaI used during the preincubation showed that the inhibitory effects of CaI on NO production could not be completely reversed by cell washing, but readdition of CaI during the 24-hour culture period produced no further inhibition of NO production (Fig. 2). Effects of other Ca-active agents on N O production. To verify that the inhibition of NO production was not limited to the use of CaI alone as a method of increasing [Ca2+]i further experiments were performed with the ionophore ionomycin and the [Ca9+]i-adenosine triphosphatase (ATPase) inhibitor thapsigargin as probes. Thapsigargin causes an increase in [Ca2+]i by

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causing a net loss of Ca 2+ from the endoplasmic reticulum Ca 2+ pool by inhibiting the ATP-driven uptake of Ca2+.19 Ionomycin promotes extracellular Ca z+ entry in a mechanism similar to CaI. As shown in Fig. 3, both ionomycin (Fig. 3, A) and thapsigargin (Fig. 3, B) produced a dose-dependent inhibition of NO accumulation at 24 hours. Neither ionomycin nor thapsigargin

induced de novo N O production in the absence of CM. Effects o f Ca-active agents on RAW cell [Ca2+]i. To confirm that CaI, ionomycin, and thapsigargin were effective in causing a rise in [Ca2+]i in our system, RAW cells from experiments in which NO determinations were performed (described above) were simultaneously assayed for [Ca2+]i levels by fluorescence spectropho-

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A. I o n o m y c i n

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Fig. 3. Effect ofionomycin and thapsigargin on NO production. Macrophage cell line RAW 264.7 (0.1 x 106 cells/well) was cultured in presence or absence of medium containing vehicle (0.1% ethyl alcohol) corresponding to highest additive concentration or CM with or without various concentrations of ionomycin (A) or thapsigargin (B) or NMA 0.5 mmol/L. Supernatants were collected at 24 hours and assayed for NO2- , Representative experiments are shown (mean -+ SEM; n = 3 per treatment group). *p < 0.01 versus CM. tometry. CaI, ionomycin, a n d thapsigargin all caused an increase in basal [Ca2+]i at concentrations identical to those resulting in decreased N O p r o d u c t i o n (Table I). Neither LPS alone, IFN-"y alone, n o r LPS + I F N ~ caused an increase in [Ca2+]i (not shown). t.Arginine d o e s not reverse the effects o f CaI on N O production. We next wished to d e t e r m i n e w h e t h e r the m e c h a n i s m o f inhibition o f NO synthesis i n d u c e d by CaI involved inhibition o f L-arginine metabolism. RAW cells were cultured for 24 hours in the presence of CM in the presence o r absence o f various concentrations of CaI o r NMA (0.5 m m o l / L ) , with increasing concentra-

tions of L-arginine. Control cultures c o n t a i n e d 0.6 m m o l / L L-arginine, which corresponds to the a m o u n t used in all previous experiments. As shown in Fig. 4, increasing concentrations of L-arginine did n o t restore N O p r o d u c t i o n that was inhibited by Cal b u t did restore N O p r o d u c t i o n that h a d b e e n inhibited by NMA to levels close to those in CM with no o t h e r additives. Similar results were seen when ionomycin or thapsigargin was substituted for CaI in these experiments (data n o t shown). T i m e course o f effects o f Ca-active agents o n N O p r o d u c t i o n . In the previous experiments the Ca-active

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T a b l e I. Effect of calcium-active agents on RAW cell

DISCUSSION

[Ca2+]i

The physiologic changes accompanying increased NO production are complex and may be at once detrimental and protective to the host. 11~ Inhibition of NO may be beneficial in thc treatmcnt of shock by reducing hypotcnsion.4, 5 In contrast, inhibition of NO synthesis in an in vivo model of hepatic injury produced microinfarcts and oxygen radical-mediatcd liver injury. 7 Inhaled NO improves pulmonary vasoconstriction after endotoxin shock, 8 and NO inhibition has been shown to worsen sepsis-induced renal hypoperfusion. 6 Some of the detrimental effects of NO production during scpsis may be mediated by other cytokines (including interleukin-6 and tumor necrosis factor--~) that arc concomitantly produced in response to release of endotoxin. 18 Although inhibition of NO synthesis by various antagonists a n d / o r inhibitors has been proposed, 4' 18 n o n e of thcse approaches has addressed the possible regulation of iNOS by endogenous intracellular mechanisms. Of the three isoforms of NOS now known to exist, the two constitutive enzymes (types I and III), which can be immediately activated to produce small amounts of NO, require Ca 2+ to alter the conformation of calmodulin, which then binds to constitutive NOS to activate NO production.3, 2o In contrast, NOS type II, present in macrophages and likely responsible for the large amounts of NO production during sepsis, is generally thought to tightly bind calmodulin without requiring Ca2+.20 This does not imply, however, that [Ca2+]i levels per se may not be important in the early signaling mechanisms during the course of cell activation by cytokines that leads to iNOS induction. We previously reported that exogenous NO donors (nitroprusside) inhibit T-cell function by reducing [Ca2+]i in both activated and resting T cells. 13 These 13 and other data 21 showed that NO may use Ca 2+ as a second messenger in T cells. Because iNOS does contain consensus sites for calmodulin binding, we hypothesized that although iNOS may not require binding of Ca 2+ to calmodulin for activation, the [Ca2+]i signaling levels achieved during cell activation may be important in the regulation of iNOS induction. In this report we present data showing for the first time that altering [Ca2+]i does indeed modify both NO end-product synthesis and iNOS mRNA induction. CaI and ionomycin both cause a rise in [Ca2+]i by promoting extracellular Ca 2+ entry into the cell, and both agents caused a dose-dependent inhibition of NO production induced by IFN-'y + LPS. To ensure that this effect was not limited to the ionophores as probes we showed that elevating [Ca2+]i with the Ca2+ATPase inhibitor thapsigargin also inhibited NO production. Thapsigargin causes a rise in [Ca2+]i predominantly by promoting its release from intracellular stores, 19 suggesting that both extracellular and intracellular Ca 2+

[Ca2+]i (nmol/L) Additive

Basal

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+ Additive

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*Results shown are from separate runs of three representative experiments. agents were present for the entire culture period after initial stimulation with CM. Because an increase in [Ca2+]i is usually regarded as an early cell activation event, we hypothesized that effects o n NO synthesis induced by changes in [Ca 2+]i may vary if [Ca 2+]i is altered at time points beyond the initial stimulation period. To test this, RAW cells were cultured as before; however, CaI, ionomycin, thapsigargin, or NMA was added at either time 0 or 4 or 8 hours after addition of CM to the culture. Supernatants were collected at 24 hours and assayed for NO2-. As shown in Fig. 5, significant inhibition of NO2- accumulation in the presence of CaI, ionomycin, or thapsigargin was not observed if these agents were added after 4 hours of culture; in contrast, inhibition by NMA was observed even if it was added up to 8 hours after culture began. Effects o f CaI on i N O S m R N A induction. The time course experiments above suggested that the effects of the Ca-active agents were likely to be generated early (4 hours or less) after stimulation by CM. We therefore wished to determine whether the inhibitory effects of CaI noted at the end-product level originated at the level of mRNA induction, because previous work has shown it to be maximally induced at 6 hours, a8 RAW cells were cultured for 6 and 24 hours in the presence of various concentrations of CaI (added at time 0) in the presence or absence of CM. Supernatant and total cellular RNA were recovered at 6 and 24 hours, respectively, and iNOS mRNA (Northern blot) and NO2- levels were determined. As shown in Fig. 6, CaI produced a dose-dependent inhibition of iNOS mRNA (Fig. 6, A) confirmed by mRNA densitometry (Fig. 6, B), accompanied by inhibition of NO end product at both 6 and 24 hours (Fig. 6, C). Similar results were obtained for ionomycin and thapsigargin (not shown).

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may be involved in the inhibition of N O observed. Providing excess L-arginine did not reverse the inhibition of N O induced by CaI, ionomycin, or thapsigargin but did restore N O production inhibited by NMA. This suggests that the mechanism of NO inhibition induced by increasing [Ca2+]i is distinct from substrate inhibition or competition. As expected, NMA inhibited N O produc-

tion even if present beyond the initial activation period, because it acts as a competitive antagonist for L-arginine metabolism by iNOS. Because adding the Ca-active agents was effective only within the first 4 hours after stimulation with IFN-',/+ LPS (but not after), we hypothesized that [Ca2+]i levels during cell activation by cytokines may regulate iNOS induction, which is maximal by

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Fig. 6. Effect of CaI on iNOS mRNA. A, Northern blot analysis of iNOS mRNA from RAW 264.7 cells cultured for 6 hours in presence of vehicle alone (0.5% dimethyl sulfoxide), CM, CM + various concentrations of CaI, or CM + 0.5% dimethyl sulfoxide. Membranes were hybridized with a complementary DNA for iNOS and then with a probe for 18s rRNA. B, Autoradiographic mRNA signals were quantitated by scanning densitometry and corrected relative to 18s rRNA. *p < 0.01 versus CM. C, NO 2- levels in culture supernatant from same RAW 264.7 cells assayed in (A) and (B) were determined after 6 and 24 hours under identical culture conditions. *p < 0.01 versus CM. 6 hours. We confirmed that CaI a n d the o t h e r Ca-active agents inhibited iNOS m R N A induction (confirmed by densitometry) in a d o s e - d e p e n d e n t m a n n e r that paralleled its effects o n e n d - p r o d u c t synthesis a n d [Ca2+]i. O t h e r r e c e n t data have suggested that regulation o f iNOS by intracellular signaling is still n o t completely understood. Thioglycollate-elicited mouse peritoneal m a c r o p h a g e s activated by IFN-',/for 20 hours exhibited a u g m e n t e d NO p r o d u c t i o n after exposure to Ca ionophores. 22 Paradoxically, however, platelet-activating factor (PAF), which also raised [Ca2+]i, h a d no effect on N O production. 22 F u r t h e r m o r e , this study assessed N O p r o d u c t i o n only after an initial 20-hour stimulation pe-

riod, b e y o n d the p o i n t where any effects o f [Ca2+]i on iNOS induction could have b e e n assessed, a n d d i d n o t examine the effects o f calcium on the initial p e r i o d o f cytokine stimulation. These data suggested that [Ca2+]i may have stimulatory effects on NOS enzyme activity well after iNOS is induced. However, we did n o t observe such effects when stimulating intracellular calcium increases after iNOS induction in o u r system. In a n o t h e r study with m u r i n e b o n e marrow--derived macrophages, [Ca2+]i was f o u n d to have absolutely no effect on N O p r o d u c t i o n i n d u c e d by LPS o r o t h e r lipopeptides. 23 Clearly, the nature o f the stimulus for N O production, the type of r e s p o n d i n g cell population, a n d timing o f the signaling events u n d e r study are i m p o r t a n t in eval-

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u a t i n g t h e r o l e o f [Ca2+]i i n t h e r e g u l a t i o n o f N O production. I n s u m m a r y , o u r d a t a s h o w t h a t [Ca2+]i is i m p o r t a n t in t h e r e g u l a t i o n o f N O p r o d u c t i o n i n R A W 264.7 cells a n d t h a t s u c h r e g u l a t i o n occurs at least as early as i N O S m R N A i n d u c t i o n . I n view o f t h e c e n t r a l role played by N O i n a variety o f i m p o r t a n t biologic functions, f u r t h e r work to clarify t h e signaling processes t h a t c o n t r o l N O synthesis in m a c r o p h a g e s a n d o t h e r cell types ks clearly indicated.

REFERENCES 1. Ochoa JB, Udekwu AD, Billiar TR, et al. Nitrogen oxide levels in patients after trauma and during sepsis. Ann Surg 1991;214: 621-6. 2. Finkel MS, Oddis CV, Jacob RD, Watkins SC, Hattler BG, Simmons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 1992;257:387-9. 3. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43: 109-tl. 4. Petros A, Bennett D, Vallance P. Effects of nitric oxide synthesis inhibitor on hypotension in patients with septic shock. Lancet 1991;2:1557-8. 5. Kilbourn RG, Gross SS, Jubran A, et al. N[G]-methyl-L-arginine inhibits tumor necrosis factor-induced hy-potension: implications for the involvement of nitric oxide. Proc Nat Acad Sci U S A 1991;84:3629-32. 6. Spain DA, Wilson MA, Garrison RN. Nitric oxide synthesis inhibition exacerbates sepsis-induced renal bypoperfusion. SURGERY 1994;116:322-31. 7. Harbrecht BG, Billiar TR, StadlerJ, et al. Inhibition of nitric oxide synthesis during endotoxemia promotes intrahepatic thrombosis and an oxygen radical-mediated hepatic injury. J Leukoc Biol 1992;52:390-4. 8. Weitzberg E, Rudehill A, Alving K, LnndbergJM. Nitric oxide inhalation selectively attenuates pulmonary hypertension and arterial hypoxia in porcine endotoxin shock. Acta Physiol Scand 1991;143:451-2; 9. Tiao G, RaffertyJ, Ogle C, FischerJE, Hasselgren P. Detrimental effect of nitric oxide synthase inhibition during endotoxemia may be caused by high levels of tumor necrosis factor and interleukin-6. SURGERY1994;116:332-8. 10. Minnard EA, ShouJ, Naama H, Cech A, Gallagher H, DalyJM. Inhibition of nitric oxide (N:O) synthesis is detrimental during endotoxemia. Arch Surg 1994;129:142-8. 11. LangrehrJM, Dull KE, OchoaJB, et al. Evidence that nitric oxide production by in vivo allosensitized cells inhibits the development of allospecific CTL. Transplantation 1992;53:63240. 12. Hoffman RA, Langrehr JM, Dull KE, McCarthy SA, Jordan ML, Simmons RL. Macrophage synthesis of nitric oxide in the moose mixed leucocyte reaction. Transplant Immunology 1994;2:31320. 13.Jordan ML, Rominski B, Hignet S, et al. Nitric oxide inhibits the T cell receptol~mediated Ca2+ second messenger system. Surg Forum 1992;43:434-6. 14. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982;126:131-7. 15.Jordan ML, Vidgen DF, Wright J, Odell M, Mills GB. Sustained increases in cystolic calcium during T lymphocyte allosensitization, proliferation, and acquisition of locomotor function. Transplantation 1991;51:464-8. 16. Chomczynski P, Sacchi N. Single-step method of RNA isolation

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by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 1987;162:156-60. 17. Lowenstein CJ, Glatt CS, Bredt DS, Snyder SH. Cloned and expressed macropbage nitric oxide synthesis contrasts with the brain enzyme. Proc Nat Acad Sci U S A 1992;89:6711-5. 18. Wang SC, Rossignol DP, Christ WJ, et aI. Suppression of lipopolysaccharide-induced macrophage nitric oxide and cytokine production in vitro by a novel lipopolysaccharide antagonist. SURGERY1994;116:339-47. 19. Thastmp O, Cullen PJ, Drobak Big, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reficulum Ca2+ATPase. Proc Natl Acad Sci U S A 1990;87:2455-70. 20. Schmidt HHHW, PonockJS, Nakane M, Forstermann U, Murad F. Ca2+/calmodulin-regulated nitric oxide synthases. Cell Calcium 1992;13:427-34. 21. Garg U, Hassid A. Nitric oxide decreases cytosolic free calcium in Balb/c 3T3 fibroblasts by a cyclic GMP-independent mechanism. J Biol CheIn 1991;266:9-12. 22. Raddassi K, Berthon B, PefitJF, Lemaire G. Role of calcium in the activation of moose peritoneal macrophages: induction of NO synthase by calcium ionophores and thapsigargin. Cell Immunol 1994;153:443-55. 23. Hauschildt S, LuckhoffA, Mulsch A, Kohler J, Bessler W, Bosse R. Induction and activity of NO synthase in bone-marrow
DISCUSSION Dr. Adrian Barbul (Baltimore, Md.). We u n d e r s t a n d fairly well the cytokine regulation of turning macrophage iNOS synthesis o n a n d off. Do cytokine effects correlate with the changes in cytoplasmic calcium levels, a n d can this explain some of the differential inhibitory or stimulatory effects of cytokine? Is calcium regulation the linchpin of the differential cytokine activity o n iNOS induction? Dr. Jordan. That is a very important question. Interferon a n d lipopolysaccharide themselves do n o t have any effects on intracellular calcium, so the effects of these cytokines in regulating calcium do n o t seem to be involved. Obviously calcium regulation can have multiple mechanisms. Reports have shown that intracellular calcium may be down-regulated or up-regulated during iNOS induction. It is very important to realize that the type of cytokine stimulation that we use, the type of the responding cell that we are studying, a n d the time that we are looking at these interactions are critical. But we have n o t seen any specific changes in iutracellular calcium after cytokine induction alone. Dr, William G. Cioffi (Providence, R.I.). Is the effect of the calcium on induction, transcription, or RNA stability? Is protein synthesis necessary in your system for induction, a n d is this where the calcium has its effect? Have you performed transcription inhibition studies to verify mRNA stability? Second, the mechanisms controlling induction and transcription of the iNOS gene are quite different in various cell types. Is the effect of calcium the same in other cell types, specifically in the vascular system? Finally, other investigators have indicated that increased calcium actually increases iNOS activity, especially when interferon--/is used as an induction agent. In one study CD53 stimulation of a macrophage line led to increased calcium a n d protein kinase activity a n d actually increased iNOS activity. How do you explain your findings in light of these data?

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Jordan et al.

Dr. Jordan. I will address the last question first. Several studies have looked at the effects of intracellular calcium levels on iNOS activity. The studies that evaluated this regulation mainly examined cells after they were induced a n d stimulated to produce cytokine. O n e study in particular that used thioglycollate-elicited macrophages stimulated t h e m for 24 hours with interferon, so the cells were already producing nitrite when the calcium reagents were added a n d an increase in nitric oxide activity occurred. Conversely, another study with bone marrow-derived macrophages by Hauschild et al. 23 found that there was absolutely n o effect of altering calcium at this late time point. Your point about stimulation with CD53 is interesting. Breakdown of phosphofipids with increase of inositol triphosphate, which stimulates intracellular calcium, is an accompanying effect of activation, but it does not imply that calcium per se is responsible for the activation. It is probably a different system when we are stimulating with lipopolysaccharide, which acts through CD14 a n d whiCh does not involve an initial increase in intracellular calcium. What we are looking at specifically is altering the levels of intracellular calcium during the activation process. As far as your second question is concerned, we have studied other macrophage cell lines and resident peritoneal macrophages with similar results. We have n o t looked at vascular endothelium or smooth muscle cells as yet. As far as the difference between induction of iNOS a n d enzyme stabilization, we are currently performing experiments studying the p r o m o t e r region of the iNOS with a luciferase reporter gene assay to determine whether we are having effects o n the p r o m o t e r region. We are also planning experiments using actinomycin D to prevent the production of further message so we can look at the stability of the message. Because

Surgery August 1995 adding our calcium-active agents beyond 4 hours h a d n o effect o n NO, we are probably not observing an effect on the stability of mRNA. Dr. Ori D. Rotstein (Toronto, Ontario, Canada). First, the stimuli for the rise in intracellular calcium that you used were not physiologic stimuli. The kinetics are different, a n d usually the height of the rise is different. Have you looked at physiologic stimuli such as PAF, for instance, that might recapitulate this effect in a more physiologic way? Second, are those reductions in mRNA expression translated into reductions in the level of iNOS protein; that is, have you specifically looked at levels of iNOS in the cell by using Western blot? Dr. Jordan. We have not specifically looked at PAF stimulation, although that would be an excellent tool to study the increase in intracellular calcium. In one published study 22 PAF was used to stimulate intracellular calcium; this was again in an assay that used macrophages 24 hours after initial stimulation with lipopolysaccharide a n d interferon. They saw n o effects o n iNOS activity with PAF, but they did see an augmentation with some of the other agents such as ionomycin, so it is very important, as you point out, to distinguish between the mechanisms by which intracellular calcium is induced. The levels of intracellular calcium that were generated in our study were within the physiologic range for these particular RAW cell fines. As far as looking at the transcripts a n d the protein, we have n o t specifically looked at induction of protein, but because we observed n o effects when the calcium agents were added later than 4 hours, we probably are not affecting the activity of the enzyme itself. However, we do have further experiments p l a n n e d to confirm this by metabolism of radioactive L-arginine a n d Western blotting, as you suggest.