Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues

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1 British Joumal of Pharmaology (1995) 114, B 1995 Stokton Press All rights reserved /95 $9. % nfluene of nitri oxide synthase inhibition, nitri oxide and hydroperoxide on insulin release indued by various seretagogues 'Georgios Panagiotidis, Bjorn Akesson, *Ewa L. Rydell & ngmar Lundquist Department of Pharmaology, University of Lund, Lund, Sweden and *Department of Pharmaology, University of Linkdping, Linkoping, Sweden 1 Reent studies have suggested that the generation of nitri oxide (NO) and hydrogen peroxide (H22) by islet NO synthase and monoamine oxidase, respetively, may have a regulatory influene on insulin seretory proesses. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presene of various pharmaologial agents known to perturb the intraellular levels of NO and the oxidation state of SH-groups. 2 The NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) dose-dependently inreased L-arginine-indued insulin release. D-Arginine did not influene L-arginine-indued insulin seretion. However, D-NAME whih reportedly has no inhibitory ation on NO synthase, modestly inreased L-arginine-indued insulin release, but was less effetive than L-NAME. High onentrations (1 mm) of D-arginine as well as L-NAME and D-NAME ould enhane basal insulin release. 3 The intraellular NO donor, hydroxylamine, dose-dependently inhibited insulin seretion indued by L-arginine and L-arginine + L-NAME. 4 Gluose-indued insulin release was inreased by NO synthase inhibition (L-NAME) and inhibited by the intraellular NO donor, hydroxylamine. Sydnonimine- (SN-1), an extraellular donor of NO and superoxide, indued a modest suppression of gluose-stimulated insulin release. SN-1 did not influene insulin seretion indued by L-arginine or the adenylate ylase ativator, forskolin. 5 The intraellular 'hydroperoxide donor' tert-butylhydroperoxide in the onentration range of.3-3 mm inhibited insulin release stimulated by the nutrient seretagogues gluose and L-arginine. Low onentrations (.3-3 gm) of tert-butylhydroperoxide, however enhaned insulin seretion indued by the phosphodiesterase inhibitor isobutylmethylxanthine (BMX). 6 slet guanosine 3':5'-yli monophosphate (yli GMP) ontent was not influened by 1mM L-arginine or tert-butylhydroperoxide at 3 or 3 gm but was markedly inreased (14 fold) by a high hydroxylamine onentration (3 gm). n ontrast, islet adenosine 3':5'-yli monophosphate (yli AMP) ontent was inreased (3 fold) by L-arginine (1 mm) and (2 fold) by tert-butylhydroperoxide (3 gm). 7 Our results strongly suggest that NO is a negative modulator of insulin release indued by the nutrient seretagogues L-arginine and gluose. This effet is probably not mediated to any major extent by the guanylate ylase-yli GMP system but may rather be exerted by the S-nitrosylation of ritial thiol groups involved in the seretory proess. Similarly the inhibitory effet of tert-butylhydroperoxide is likely to be eliited through affeting ritial thiol groups. The mehanism underlying the seretionpromoting ation of tert-butylhydroperoxide on BMX-indued insulin release is probably linked to intraellular a2'-perturbations affeting exoytosis. 8 Taken together with previous data the present results suggest that islet prodution of low physiologial levels of free radials suh as NO and H22 may serve as important modulators of insulin seretory proesses. Keywords: solated islets; insulin release; L-arginine; N-nitro-D-arginine methyl ester; N-nitro-L-arginine methyl ester; D-arginine; hydroxylamine; tert-butylhydroperoxide; gluose; yli GMP; yli AMP ntrodution Synthesis of nitri oxide (NO) from L-arginine has been is a negative modulator of L-arginine-indued insulin release identified as an important mehanism impliated in the (Panagiotidis et al., 1992). The insulinotropi ation of L- regulation of ellular funtion and ommuniation (see Mon- arginine is omplex and has partially been attributed to a ada et al., 1991). Very reently, evidene has been obtained diret depolarizing effet of the ationi amino aid. Sine for the ourrene of NO synthase ativity in both endorine NO synthase inhibition inreased L-arginine-indued insulin ells and nerves of the islets of Langerhans (Shmidt et al., release (Panagiotidis et al., 1992) we suggested that the 1992; Vinent, 1992; Panagiotidis et al., 1992; orbett et al., stimulating effet of L-arginine was in part ounterated by 1993). Previous data (Layhok et al., 1991; Shmidt et al., the onomitant prodution of NO. n other tissues the 1992) suggested that the islet NO-system had a promoting L-arginine-derived NO is thought to at prinipally by ation on insulin seretion. n ontrast, however, we reently stimulating guanylate ylase, resulting in the elevation of presented in vitro results showing that the NO system in fat guanosine 3':5'-yli monophosphate (yli GMP) (Monada et al., 1991). With regard to insulin sereting mehanisms we reently hypothetised (Panagiotidis et al., 'Author for orrespondene. 1992) that the inhibitory ation of NO might be exerted

2 29 through an interation of NO with thiol groups essential for the seretory proess. n fat, it has been known for a long time that ertain membrane thiols are involved in the stimulus-seretion oupling of gluose-indued release of insulin (see Hellman et al., 1974; Ammon & Mark, 1985). Thus the formation of S-nitrosothiols may hange the balane of ritial intraellular and/or membrane thiols of the P-ells. Suh a mehanism is in lose agreement with our reent proposal that hydrogen peroxide (H22) generated by the ativation of islet monoamine oxidase may inhibit ertain insulin sereting proesses by oxidizing ritial thiol groups (Lundquist et al., 1991; Panagiotidis et al., 1993b,). The aim of the present investigation was to study in more detail the effet of NO and 'H22' (as mimiked by tertbutylhydroperoxide) on insulin seretion indued by various seretagogues. This was aomplished by means of the NO synthase inhibitor, N-nitro-L-arginine methylester (L- NAME), the intraellular NO donor hydroxylamine (De Master et al., 1989), the extraellular donor of NO and superoxide sydnonimine-1 (SN-1) (Hogg et al., 1992) as well as the intraellular 'hydroperoxide donor' tert-butylhydroperoxide (Orrenius & Moldeus, 1984). Methods Animals Female mie of the NMR strain (B & K, Universal, Sollentun Sweden) weighing 25-3g, were used throughout the experiments. A standard pellet diet (ASTRA-Ewos, S6dertalje, Sweden) and tap water were available ad libitum before all experiments. Drugs and hemials N-nitro-L-arginine methyl ester (L-NAME), D-NAME, L- and D-arginine, 3-isobutyl-l-methylxanthine (BMX) and hydroxylamine were from Sigma hemials, St Louis, MO, U.S.A. tert-butylhydroperoxide was purhased from Merk AG, Darmstadt, Germany. ollagenase (LS4) was obtained from Worthington Biohemial orp., Freehold, NJ, U.S.A. Bovine serum albumin was from N Biomedials Ltd., High Wyombe, Buks, U.K. Sydnonimine- (SN-1) was kindly supplied by GEA Ltd., Pharmaeutial Researh Laboratories, DK-265 Hvidovre, Denmark. The radioimmunoassay kits for insulin determination were obtained from NOVO Ltd., Bagsvaerd, Denmark. The antibodies for yli GMP and yli AMP determination were prepared in our laboratory. The preparation and haraterization of the antiserum were made aording to Steiner et al. (1972). ['25lguanosine or [1251]adenosine 3':5'-yli phosphori aid was from NEN Dupont, Dreieh, Germany. All other drugs and hemials were from British Drug Houses Ltd., Poole, Dorset, U.K. or Merk AG., Darmstadt, Germany. Experimental protool Preparation of isolated islets from the mie was performed by retrograde injetion of a ollagenase solution via the bilepanreati dut (Gotoh et al., 1985). Freshly isolated islets were preinubated in a HEPES-supplemented KRB-buffer as previously desribed (Panagiotidis et al., 1992). After preinubation the buffer was hanged and the islets were inubated with the test agents for 6 min at 37 in an inubation box (3 yles min-') if not otherwise stated. The inubation vials ontained 8 islets per 1.5 ml buffer exept when islets were inubated for yli nuleotide determination where 75-8 islets per vial were needed. mmediately after inubation an aliquot of the medium was removed and frozen for subsequent assay of insulin. For analysis of islet yli GMP and yli AMP the inubation was stopped by the removal of buffer and addition of.5 ml ie-old 1% trihloroaeti G. Panagiotidis et al Effet of NO and hydroperoxide on insulin release aid (TA), followed by immediate freezing in a - 7 ethanol bath. When assayed,.5 ml H2 was added and the samples were soniated for 3 x 5 s in a Branson Sonifier ell disrupter followed by entrifugation at 1.1 g for 15 min. The supernatants were olleted and extrated with 4 x 2 ml of water-saturated diethyl ether. The aqueous phase was reovered and freeze dried, using a Lyova GT 2 freeze dryer. The residue was then dissolved in 45 Ll 5 mm Naaetate buffer (ph 6.2). Both yli GMP and yli AMP were determined by radioimmunoassay aording to Steiner et al. (1972). n order to improve sensitivity of the assays, the samples were aetylated with aeti aid anhydride as desribed by Harper & Brooker (1975). Results Effet of NO synthase inhibition on basal and stimulated insulin seretion Figure la shows the effet of different onentrations of the NO synthase inhibitor L-NAME (an L-arginine analogue) on basal insulin seretion (7 mm gluose) from isolated islets. L-NAME-onentrations of.1-5.mm did not signifiantly influene basal insulin release. A high onentration of L- NAME (1 mm), however, indued a 3 fold inrease in insulin seretion. Figure lb and l illustrate the influene of NO synthase inhibition on insulin release stimulated by L-arginine and gluose, respetively. onentrations of L-NAME whih did not affet basal insulin seretion (Figure l dosedependently potentiated L-arginine-indued insulin release (Figure lb). Furthermore, NO synthase inhibition did also inrease gluose-stimulated insulin seretion (Figure l). Effet of D-arginine and D-NAME on basal and L-arginine-stimulated insulin seretion Figure 2a ompares the effets of L-arginine and D-arginine, the stereoisomer of L-arginine, on insulin release. Similar to 5 mm L-NAME (Figure l 5 mm D-arginine had no insulin releasing effet (Figure 2. However, unlike L-NAME, D- arginine did not influene L-arginine-indued insulin release. Figure 2b shows that D-NAME, whih similar to D-arginine, reportedly is devoid of NO synthase inhibitory properties modestly inreased L-arginine-indued insulin release but to a signifiantly lower level than did L-NAME. A high onentration of D-arginine (1 mm) (Figure 2, however, indued a moderate insulin response whih was approximately 4 times less than that of 1 mm L-arginine (Figure 2b). D-NAME (1 mm) ould stimulate basal insulin release to the same extent as L-NAME (Figure 2b). Effet of the intraellular NO donor, hydroxylamine, on the seretion of insulin indued by L-arginine and gluose Figure 3 illustrates the effet of the intraellular NO donor hydroxylamine on insulin seretion indued by 1 mm L- arginine in the presene of 7 mm basal gluose (Figure 3. Figure 3b shows the effet of hydroxylamine on insulin release stimulated by 16.7 mm gluose. The NO donor dosedependently inhibited L-arginine stimulated insulin release whih was almost totally suppressed at 3 mm hydroxylamine. Gluose-indued insulin release appeared less sensitive to the drug, although a total suppression was again ahieved at 3 mm hydroxylamine. Hydroxylamine did not influene basal (7 mm gluose) insulin seretion (data not shown). Effet of the extraellular NO and superoxide donor SN- on insulin seretion indued by gluose, L-arginine andforskolin The next series of experiments examined the effet of the NO and superoxide donor SN-1, whih is believed to release NO

3 and superoxide extraellularly. Figure 4a shows that SN- signifiantly redued insulin release stimulated by 16.7 mm gluose. This inhibition was modest and detetable already at.3 plm SN- but was less pronouned than the inhibitory effet of hydroxylamine (Figure 3b). SN- had no influene on insulin seretion indued by L-arginine (Figure 4b) or by the adenylate ylase ativator, forskolin (Figure 4). Note that the SN-1 experiments were run only for 35 min beause of the reported rapid deterioration of this ompound. Effet of the intraellular 'hydroperoxide donor' tert-butylhydroperoxide on insulin seretion indued by L-arginine, gluose and isobutylmethylxanthine (BMX) 4). 1 4._ () L. n analogy with the experiments on intraellular delivery of NO by hydroxylamine we then examined the effet of intraa 4, 2- O b G. Panagiotidis et al Effet of NO and hydroperoxide on insulin release L-NAME (mm) P<.2 l ' 14 s ~~~~~* W 12 'U; 1 8 _ 6-4 _.1 L-NAME (mm) Figure 1 ( Effet of different onentrations of NG-nitro-L-arginine methyl ester (L-NAME) on basal insulin seretion (7 M gluose) from isolated islets. Values are mean ± s.e.mean from 6-1 bathes of islets **P<.1 vs basal ontrol. (b) nfluene of different onentrations of L-NAME on insulin release from isolated islets stimulated by L-arginine 5 mm in the presene of basal gluose 7 mm (solid olumns). Gluose 7 mm basal ontrol is denoted by an open olumn. Values are mean ± s.e.mean from 6-1 bathes of islets, *P<.5, **P<.1, ***P<O.OO1 ompared to 5mM L-arginine without addition of L-NAME. () Gluose-indued (16.7 mm) insulin release from isolated islets in the absene (open olumn) or presene (solid olumn) of 5 mm L-NAME. Values are mean ± s.e.mean from 6-1 bathes of islets, *P<.5. 5 ellular hydroperoxide by employing tert-butylhydroperoxide. Data from previous in vivo experiments suggested that an intraellular generation of H22 by the ativation of islet monoamine oxidase might impair gluose-indued but promote BMX-indued insulin release (Lundquist et al., 1991; Panagiotidis et al., 1993b,, 1994). Figure 5a and b illustrate the effet of tert-butylhydroperoxide on insulin seretion indued by 1mM L-arginine and 16.7mM gluose, respetively. The 'hydroperoxide donor' markedly suppressed both L-arginine- and gluose-indued insulin release. n ontrast insulin seretion indued by BMX, an ativator of the yli AMP system, was enhaned by low levels of tert-butylhydroperoxide (.3-3 jlm) (Figure S). A very high onentration of tert-butylhydroperoxide (3 mm) suppressed BMXindued insulin release. tert-butylhydroperoxide did not influene basal (4 mm gluose) insulin seretion (data not shown). omparative effets of hydroxylamine and tert-butylhydroperoxide on insulin seretion indued by L-arginine + L-NAME n order to examine the effet of hydroxylamine and tertbutylhydroperoxide on L-arginine-indued insulin seretion in the absene of endogenous NO prodution, a series of experiments was performed with the NO synthase inhibitor L-NAME. Figure 6a shows that hydroxylamine dose-dependently suppressed L-arginine-indued insulin release in the presene of L-NAME with a total inhibition at 3 mm. Figure 6b shows that tert-butylhydroperoxide was less potent in this respet displaying an inhibitory ation of approximately 5% n. 2- m 1- - t- b 5-4 o._ 3-2- '- mlls~~~~~~. *** ~ ~ ^~~~~~ -T.** P<.1 i Ị ,. X rt*h P<.5 m Figure 2 ( Effet of L-arginine 5 mm (solid olumn), D-arginine 5 mm (ross-hathed olumn), L-arginine 5 mm + D-arginine 5 mm (stippled olumn), L-arginine 1 mm (hathed olumn) and D- arginine 1 mm (striped olumn) on insulin seretion from isolated islets in the presene of 7 mm gluose. Basal ontrol (7 mm gluose) is denoted by an open olumn. Values are mean ± s.e.mean from 6-1 bathes of islets, **P<.1, ***P<.1 ompared to basal ontrol. (b) Effet of L-arginine 5 mm (solid olumn), L-NAME 1mm (ross-hathed olumn), D-NAME 1mm (stippled olumn), L-arginine 5 mm + L-NAME 5 mm (hathed olumn) and L-arginine 5 mm + D-NAME 5 mm (striped olumn) on insulin seretion from isolated islets in the presene of 7 mm gluose. Basal ontrol (7 mm gluose) is denoted by an open olumn. Values are mean ± s.e.mean from 6-1 bathes of islets, ***P<.1, ompared to L-arginine 5mM. *** 291

4 292 G. Panagiotidis et a/ Effet of NO and hydroperoxide on insulin release of the L-arginine + L-NAME insulin response at.3 and 3 mm. Effet of L-arginine, hydroxylamine and tert-butylhydroperoxide on islet yli GMP and yli AMP levels Table 1 shows the effet of 1 mm L-arginine as well as onentrations of 3 and 3 jlm hydroxylamine and tertbutylhydroperoxide on the levels of yli GMP and yli AMP in isolated mouse islets at 7 mm gluose. The gluose onentration in the ontrol inubation medium was 7 mm. t is seen that only 3 tlm hydroxylamine influened the yli GMP levels. At this onentration the NO donor evoked a 14 fold inrease in islet yli GMP ompared to ontrol levels. n ontrast, the yli AMP levels were inreased by L-arginine as well as by the 3 jlm onentration of tertbutyl-hydroperoxide (Table 1). Disussion The present results show that the L-arginine-analogues L- NAME (an NO synthase inhibitor), and D-arginine (whih does not generate NO), ated differently on insulin release when inubated together with L-arginine. nreasing onentrations of L-NAME (.1-5. mm), whih did not influene basal insulin release, indued a dose-related potentiation of the insulin response evoked by a submaximal onentration of L-arginine (5 mm). D-Arginine (5 mm), however, did not influene L-arginine-indued insulin release. On the other, 3. 1) L U (a L- 4a hand D-NAME (5 mm) whih reportedly is devoid of NO synthase-inhibitory properties (Radomski et al., 199; Rees et al., 199) also inreased the insulin release indued by L- arginine but was less effetive than L-NAME. Moreover, a high onentration (1 mm) of L-NAME, D-NAME or D- arginine ould per se indue insulin seretion, D-arginine being less effiient in this respet. The data suggest that the observed effets of the L-arginine analogue L-NAME, at least partially, ould be asribed to its ationi properties. However, the fat the D-NAME was less effetive than L-NAME in inreasing L-arginine-indued insulin release strongly suggests that part of the effet was due to NO synthase inhibia b 3- P<.1 T T ** ** FE1F'f SN-1 (gm), o L- L -T o. 2 1* a o L- ) Hydroxylamine (gm) -.3 P < D 1.- a ) ,..2-7T SN-1 (gm) -r Hydroxylamine (gm) Figure 3 ( Effet of different onentrations of hydroxylamine on insulin release from isolated islets stimulated by L-arginine 1 mm in the presene of basal gluose 7 mm (solid olumns). Gluose 7 mm basal ontrol is also shown (open olumn). Values are mean ± s.e.mean from 9 bathes of islets, *P<.5, ***P<.1 ompared to 1 mm L-arginine without addition of hydroxylamine. (b) Effet of different onentrations of hydroxylamine on gluoseindued insulin seretion (16.7 mm gluose) from isolated islets. Values are mean ± s.e.mean from 9 bathes of islets, **P<.1. en -.3 SN-1 (gm) Figure 4 ( Effet of different onentrations of sydnonimine-l (SN-) on gluose indued insulin seretion (16.7 mm gluose) from isolated islets during a 35 min inubation period. Values are mean ± s.e.mean from 9 bathes of islets, *P<.5, **P<O.O ompared to 16.7 mm gluose without addition of SN-. (b) Effet of SN- on L-arginine-stimulated (1 mm) insulin release in the presene of 7 mm gluose (solid olumns). Gluose 7 mm basal ontrol is also shown (open olumn). Values are mean ± s.e.mean from 7 bathes of islets. () Effet of SN-1 on insulin release from isolated islets stimulated by forskolin (2 gm) in the presene of 4 mm gluose (solid olumns). Gluose 4 mm basal ontrol is also shown (open olumn). Values are mean ± s.e.mean from 9 bathes of islets. 3

5 tion. t should be noted, however, that the stereoisomer D-arginine, whih arries the same ationi properties as L-arginine and does not generate NO, was a weaker insulin seretagogue than L-arginine even in the presene of L- NAME. This suggests that other unknown fators may ontribute to the differene in the insulin releasing ation of D- and L-arginine. One explanation might be the existene of different transport mehanisms aross the plasma membrane ) L- 8- z 7, 6-5 ) a -4. a 3- LO 2- =: 1- - G. Panagiotidis et al Effet of NO and hydroperoxide on insulin release " -7- a 6- -.D 5- D P<.2 ad 3- T - a l 2-1. _' 1 - a tert-butylhydroperoxide b (gm) 5- - b 7 P<.1. _ ) 3 2- &- * 1- ** T -.L rri Figure 6 tert-butylhydroperoxide (gm) * ) ' 2-? 1- n o - P< ( r- ) ** n.s tert-butylhydroperoxide (gm) Figure 5 ( Effet of different onentrations of tert-butylhydroperoxide on insulin release from isolated islets stimulated by L-arginine 1O mm in the presene of basal gluose 7 mm (solid olumns). Gluose 7 mm basal ontrol is also shown (open olumn). Values are mean ± s.e.mean from 6-9 bathes of islets, *P <.5, **P<.1 ompared to 1O mm L-arginine without addition of tertbutylhydroperoxide. (b) Effet of different onentrations of tertbutylhydroperoxide on gluose-indued insulin seretion (16.7 mm gluose) from isolated islets. Values are mean ± s.e.mean from 6 bathes of islets, *P<.5, **P<.1 ompared to 16.7 mm gluose without addition of tert-butylhydroperoxide. () Effet of different onentrations of tert-butylhydroperoxide on insulin release from isolated islets stimulated by isobutylmethylxanthine (BMX) 1 mm in the presene of basal gluose 4 mm (solid olumns). Gluose 4 mm basal ontrol is also shown (open olumn). Values are mean± s.e.mean from 9 bathes of islets, *P<.5, **P<O.O ompared to 1 mm BMX without addition of tertbutylhydroperoxide. as previously shown in endothelial ells (Bogle et al., 1992). Thus, D-arginine might not be aumulated as rapid as or to the same extent as L-arginine and L- or D-NAME. However, taken together our data indiate that L-arginine has a dual a P<.2 ** T** Hydroxylamine (gm) P<.1 m * * tert-butylhydroperoxide (gm) ( nfluene of different onentrations of hydroxylamine on insulin release from isolated islets stimulated by L-arginine 5 mm + NO-nitro-L-arginine methyl ester (L-NAME) 5 mm in the presene of basal gluose 7 mm (ross-hathed olumns). Effets of basal gluose (7 mm) (open olumn), and L-arginine 5 mm (solid olumn) are also shown. Values are mean ± s.e.mean from 6-9 bathes of islets, **P<.1, ***P<.1 ompared to 5mM L- arginine+l-name without addition of hydroxylamine. (b) nfluene of different onentrations of tert-butylhydroperoxide on insulin release from isolated islets stimulated by L-arginine 5 mm + L- NAME 5 mm in the presene of basal gluose 7 mm (ross-hathed olumns). Effets of basal gluose (7 mm) (open olumn), and L- arginine 5 mm (solid olumn) are also shown. Values are mean ± s.e.mean from 6-9 bathes of islets, *P <.5 ompared to 5 mm L-arginine + L-NAME 5 mm without addition of tertbutylhydroperoxide. Table 1 Aumulated levels of yli GMP and yli AMP in isolated islets after a 6 min inubation period in the presene of 1 mm L-arginine or different onentrations of hydroxylamine and tert-butylhydroperoxide ontrol L-Arginine 1 mm Hydroxylamine 3 tmm Hydroxylamine 3 tim tert-butylhydroperoxide 3 jm tert-butylhydroperoxide 3 tmm yli GMP yli AMP (amol/islet) (fmol/islet) 333 ± ± ± ± 95*** 481 ± ± ± ± 3.12** 9.66 ± ± ± ±.71 * Basal medium (ontrol) ontained 7 mm gluose +.2 mm BMX. Means ± s.e.mean from 5-7 bathes of islets. Asterisks (*) denote signifiant differene vs ontrol. *P <.2; **P<.5; ***P<

6 294 ation on insulin release as previously suggested (Panagiotidis et al., 1992). Thus the ationi depolarizing ation of L- arginine promotes insulin seretion, whereas the NO derived from L-arginine metabolism negatively modulates the seretory proess. The results from previous investigations with rat islets or ells from the lonal panreati P-ell line, HT-T5, as well as RNm5F insulinoma ells using the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA) (Layhok et al., 1991; Shmidt et al., 1992) suggested that NO enhanes L-arginine-stimulated insulin release. The reason for this disrepany in results is presently unlear. However, the P-ell lines may have a different reation pattern ompared with normal islets. Further, we reently observed that during our inubation onditions L-arginine-indued insulin release also from rat islets was enhaned by NO synthase inhibition (data not shown). These results are also ompatible with previous data on the effet of L-NMMA on insulin release from ultured rat islets (Jansson & Sandler, 1991). Moreover, in ontrast to previous reports (Layhok et al., 1991; Shmidt et al., 1992; Jones et al., 1992) our present results suggest that gluose-indued insulin seretion (16,7 mm gluose) is inreased in the presene of NO synthase inhibition. This may suggest that L-NAME inhibited a basal 'tone' of NO generation within the isolated islets in the presene of high gluose and/or that L-NAME exerted a ationi potentiation of gluose-indued insulin seretion. The fat that 1 mm of either sterioisomeri form of NAME alone released the same amounts of insulin favours the latter assumption. Thus, to study in more detail the inhibition of NO synthase ativity in islet P-ells, a new generation of NO synthase inhibitors has to be developed that hopefully does not share the ationi properties of the L-arginine analogues. n aordane with our suggestion that NO in fat is a negative modulator of L-arginine-indued insulin seretion, the intraellular NO donor, hydroxylamine, displayed a dosedependent inhibition of the insulin response to L-arginine alone and to the ombined ation of L-NAME + L-arginine. Moreover, gluose-indued insulin seretion was also markedly inhibited by intraellular NO generation indued by hydroxylamine. Hydroxylamine is a known preursor of NO in biologial systems and it has even been suggested that it may be an intermediate in the onversion of L-arginine to NO (DeMaster et al., 1989). Further, previous data have suggested that hydroxylamine easily penetrates into ells where it is oxidized to NO and exerts its effets. atalase is known to atalyze this reation (DeMaster et al., 1989). With regard to SN-1 the effet is probably in the interstitial environment as, from its hemial struture, this ompound is not likely to gain aess to the ell interior. Hene the 'mixed' free radials, NO and superoxide, generated by SN-1 (Hogg et al., 1992) may at mainly extraellularly. t has previously been shown that diret oxidation of 1-ell superfiial thiols lead to a2+ uptake and subsequent inrease of insulin seretion at low gluose (Ammon & Mark, 1985). However, oxidation of intraellular thiols inhibits gluoseindued insulin seretion (Ammon & Mark, 1985). Thus the ation of SN-1 is very omplex, sine this ompound generates both superoxide and NO, whih then an at both extra- and intraellularly. Apparently the intraellular effet of the evolved NO and superoxide dominates with regard to gluose-indued insulin release, whereas insulin release stimulated by L-arginine is unaffeted (balane between extraellular and intraellular effets of free radials?). Similarly forskolin-indued insulin release was unaffeted by SN-1 even at a very low gluose onentration (yli AMP is known to lower the threshold for gluose-indued insulin release) onfirming earlier data showing that ativation of the yli AMP system by (-)-isoprenaline is not affeted by thiol oxidation (Ammon & Mark, 1985). Therefore, it should be noted that although the main effet of NO has been asribed to the intraellular ativation of guanylate ylase, G. Panagiotidis et al Effet of NO and hydroperoxide on insulin release reent observations have suggested that NO might exert its ation also through yli GMP-independent events (Stamler et al., 1992). Thus S-nitrosylation of ritial thiol groups on e.g. enzyme and reeptor proteins has been suggested as a potential mehanism for modifiation of proteins with important regulatory funtions (Stamler et al., 1992). The ombined data on the effets of L-NAME on one hand and the intraellular NO donor hydroxylamine on the other thus strongly suggest that NO is a negative modulator of insulin seretion indued by L-arginine and gluose. However, it should be noted that up to date biohemial evidene for the presene of a onstitutive NO synthase in islet tissue is restrited to the report of Shmidt et al. (1992) who used HT T15 lonal panreati P-ells. Measurement of nitrite prodution as an indiator of NO synthase ativity has been used by several authors (f. Monada et al., 1991; orbett et al., 1993). However, this may be of limited value in shortterm inubation of islet tissue, sine aording to our hypothesis muh of the NO produed is 'trapped' by S- nitrosylation, (see also Stamler et al., 1992). Moreover, omparatively high onentrations of NO synthase inhibitors (in the order of M) have been used to detet an inhibitory ation on nitrite prodution from ultured islets (Welsh et al., 1991; orbett et al., 1993). Therefore, a more detailed biohemial haraterization of islet NO synthase(s) is needed. On the other hand, the immunohistohemial evidene for the ourrene of a onstitutive NO synthase in islet 1-ells seems to be very strong, sine this has been demonstrated with three different antisera (Shmidt et al., 1992; orbett et al., 1993). Moreover, islets ultured with.5 mm L-NMMA displayed a substantially redued NADPH diaphorase staining (orbett et al., 1993). With regard to the inhibition of NO synthase it ould be argued, however, that we used a omparatively high onentration of L-NAME (5 mm) (Monada et al., 1991; F6rstermann et al., 1991) in the L-arginine vs. L-NAME interation experiments. Sine the inhibiting ation of L-NAME is ompetitive (Monada et al., 1991) and sine onentrations of 5-1 mm L-arginine are needed to obtain a reliable insulin release (Gerih et al., 1974) it was neessary to use the highest onentration of L-NAME that did not per se affet insulin release (f. Figure l İt has been known for a long time (see Hellman et al., 1974; Ammon & Mark, 1985) that superfiial thiols are of importane for ertain insulin seretagogues, and that the redued state of the intraellular GSH/GSSG system, whih maintains the superfiial thiols in the redued state, is a prerequisite for gluose-stimulated a2+ uptake and insulin release. Hene it is not inoneivable that NO-indued S- nitrosylation may impair the funtion of ritial thiol groups of importane for insulin seretion indued by gluose and L-arginine. This might be aomplished either diretly or as a onsequene of deranging the balane of the GSH/GSSG system. Our present finding that L-arginine-indued insulin release is more sensitive to NO-inhibition than insulin seretion stimulated by gluose is in good agreement with previous observations (Ammon & Mark, 1985) showing that gluose metabolism inreases the GSH/GSSG ratio by generating NADPH from the pentose shunt. Similar to the desribed effets of NO prodution, it has been proposed that the H22 evolution indued by the ativation of islet monoamine oxidase ativity negatively modulates gluose-indued insulin seretion by impairing the funtion of ritial thiol groups (Lundquist et al., 1991; Panagiotidis et al., 1993b,). The present results, whih show that the intraellular 'hydroperoxide donor' tert-butylhydroperoxide inhibited insulin seretion indued by gluose and L-arginine strongly support suh an assumption. n fat the pattern of insulin seretory suppression indued by tert-butylhydroperoxide ('H22') and hydroxylamine (NO) was remarkably similar, although on a molar basis NO appeared more potent in this respet. t should be emphasized, however, that although both hydroxylamine and tert-butylhydroperoxide

7 are believed to exert their effets intraellularly, the physiologial ativation of NO synthase (NO) and monoamine oxidase (H22) may indue a muh more loalized inrease of these messengers, whih makes preise omparisons diffiult. Moreover, it is not finally established that tert-butylhydroperoxide has exlusively intraellular ations (Orrenius & Moldeus, 1984). Reent in vivo studies have suggested that ativation of islet monoamine oxidase by ertain yli AMP-inreasing agents suh as the P2-adrenoeptor agonist, terbutaline (Panagiotidis et al., 1993b,) and the phosphodiesterase inhibitor BMX (Panagiotidis et al., 1994) ould failitate the insulin releasing ation of these agents by degrading inhibitory monoamines and produing small levels of exoytosispromoting H22. n ontrast to insulin release stimulated by nutrients suh as gluose and L-arginine, the seretory proess indued by yli AMP ativating agents seem to operate largely independently of the GSH/GSSG balane as well as of extraellular a2+ (Ammon & Mark, 1985; Draznin, 1988; Salehi & Lundquist, 1993). The present in vitro results, whih show that very small levels (.3-3 gm) of the intraellular 'hydroperoxide donor' tert-butylhydroperoxide indeed enhaned BMX-indued insulin release from isolated islets are in good agreement with our hypothesis. The detailed mehanism of ation of this failitation of BMX-indued insulin release by hydroperoxide remains to be eluidated. However, it is known from other tissues that peroxides an promote granule fusion with membranes, and that H22 prodution an result in perturbation of intraellular a2' homeostasis leading to an inrease in ytosoli free a2+ onentration (reutz, 1981; Orrenius & Moldeuis, 1984). Finally, a onentration of L-arginine (1 mm) whih indued a large stimulation of insulin release in the presene of a physiologial onentration of gluose (7 mm) did not affet islet yli GMP levels (Table 1). These results are in aordane with a reent study (Jones et al., 1992) and do not suggest that L-arginine-derived NO initiates insulin seretion through inreasing islet yli GMP ontent as previously proposed (Layhok et al., 1991). n fat, the NO donor, hydroxylamine (at a onentration of 3 LM) indued a marked inrease in islet yli GMP levels. This onentration of hydroxylamine almost totally suppressed insulin seretion indued by gluose or L-arginine. tert-butylhydroperoxide on the other hand did not signifiantly influene the islet yli GMP ontent. Hene, most evidene thus far suggests that NO is a negative modulator of gluose- and L-arginine-indued insulin release and that yli GMP (at least at very high levels) may even ontribute to this suppressive ation (see also Vara & Tamarit-Rodriguez, 1991). G. Panagiotidis et al Effet of NO and hydroperoxide on insulin release 295 With regard to islet yli AMP we were able to show that 1 mm L-arginine brought about an inrease of this nuleotide in islet tissue. This novel finding suggests that raised yli AMP levels may ontribute to the insulin releasing ation of L-arginine. However, it annot be exluded that effets on yli AMP in the gluagon ells may be involved. Similarly, the stimulating effet of tert-butylhydroperoxide (3 jam) on islet yli AMP levels does not orrelate to insulin release and thus may reflet either yli AMP hanges in the gluagon ells or simply illustrate the notion that yli AMP itself is not an initiator of insulin release (Ammon & Mark, 1985). n onlusion, suppression and enhanement of NO generation by the NO synthase inhibitor, L-NAME, on the one hand, and the intraellular NO donor hydroxylamine on the other, indued paralell hanges in insulin release stimulated by L-arginine and gluose. These findings are ompatible with NO being a negative modulator of the seretory events. The main effet of NO is probably not exerted through elevated yli GMP levels but rather through S-nitrosylation of ertain thiol groups ritial for the seretory proess. Suh an assumption is strengthened by the observation that the intraellular 'hydroperoxide donor' tertbutylhydroperoxide, a reognized thiol oxidant, indued almost similar hanges in the insulin releasing pattern. yli AMP ativating seretagogues suh as forskolin and BMX appeared muh less sensitive to the inhibitory ation of NO and hydroperoxide. n ontrast, small levels of hydroperoxide were found to enhane BMX-indued insulin release. Thus the present data showing a differential in vitro effet of tert-butylhydroperoxide on insulin seretion indued by gluose and BMX, respetively, are in good agreement with the results of previous in vivo experiments (Lundquist et al., 1991; Panagiotidis et al., 1993b,) suggesting that the H22 generated by ativation of islet monoamine oxidase ativity inhibits gluose-indued insulin seretion and promotes insulin release stimulated by BMX. 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