Influence of K⁺-induced depolarization on carbachol-L-NAME interactions on islet hormone release

Recently it was reported (Krippeit-Drews et al. 1996) that L-NAME in concentrations >5 mM might bring about closure of the ATP-sensitive K⁺ channels (K_ATP channels) in the -cell membrane. In the present experiment we therefore depolarized the membrane with high K⁺ (30 mM) in the presence of 250 µM diazoxide (keeping the K_ATP channels open) (Gembal et al. 1992) to elucidate whether NOS inhibition would still affect islet hormone release. Figure 5 shows that L-NAME, also in this experimental situation, markedly enhanced carbachol-induced insulin release and inhibited glucagon release. However, it should be noted that the potentiating effect by L-NAME on carbachol-stimulated insulin release in depolarized islets was less pronounced when compared with the potentiating effect in normal, non-depolarized islets. This is suggestive of a minor non-specific action by 5 mM L-NAME probably involving a slight effect on K_ATP channels (Krippeit-Drews et al. 1996) during carbachol stimulation. In K⁺-diazoxide-treated control islets in the absence of carbachol, NOS inhibition by L-NAME had no effect on insulin release but slightly suppressed glucagon release (Fig. 5).

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Figure 5. Effect of L-NAME on carbachol-induced islet hormone release in depolarized islets Effect of NG-nitro-L-arginine methyl ester (L-NAME) on insulin and glucagon release stimulated by carbachol (50 µM) in the presence of 30 mM K+, 250 µM diazoxide and 7 mM glucose (). Basal controls (), 30 mM K+ and 250 µM diazoxide controls () and carbachol controls () are included. Values are means ± S.E.M. from 10-24 incubation vials containing 10 islets each. The incubation period was 60 min. Each experiment comprised 4-5 incubation vials for each of the 4 test groups. * P < 0·05, ** P < 0·01, *** P < 0·001.

Influence of the intracellular NO donor hydroxylamine on insulin and glucagon release stimulated by carbachol

To test whether a pharmacologically induced intracellular increase in the evolution of NO would influence islet hormone release the intracellular NO donor hydroxylamine was employed. Figure 6A shows that hydroxylamine dose-dependently inhibited carbachol-induced insulin release in the presence of 7 mM basal glucose. Carbachol-stimulated glucagon secretion, in contrast, was enhanced but only at a low concentration of hydroxylamine (3 µM) and was unaffected by higher concentrations (Fig. 6B).

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Figure 6. Effects of hydroxylamine on carbachol-induced islet hormone release Effect of different concentrations of the NO donor hydroxylamine on insulin and glucagon release stimulated by carbachol (100 µM). Values are means ± S.E.M. of 6-8 incubation vials on each point. Each vial contained 8 islets. The incubation period was 60 min. * P < 0·05; ** P < 0·01; *** P < 0·001.

In vivo effect of L-NAME on carbachol-stimulated insulin and glucagon response

Figure 7A shows that the insulin secretory response to an intravenous injection of carbachol was greatly enhanced by a previous injection of L-NAME. The peak plasma insulin levels after carbachol were 942 ± 120 vs. 2574 ± 210 pmol l^-1 (L-NAME + carbachol) (P < 0·001). In contrast, the carbachol-induced glucagon response was suppressed by L-NAME (Fig. 7B). The peak plasma glucagon levels following carbachol injection were 1048 ± 98 vs. 521 ± 52 ng l^-1 (L-NAME + carbachol) (P < 0·001). Basal plasma levels of insulin and glucagon were not significantly influenced by the NOS inhibitor (Fig. 7).

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Figure 7. In vivo action of L-NAME on carbachol-stimulated insulin and glucagon responses In vivo effect of NG-nitro-L-arginine methyl ester (L-NAME) pretreatement on the acute insulin and glucagon secretory response to an I.V. injection of carbachol (0·16 µmol kg-1). L-NAME (1·2 mmol kg-1) was administered I.V. 15 s prior to carbachol. Basal controls showing plasma hormone levels after saline + saline and L-NAME + saline are included. Values are means ± S.E.M. from 12-17 animals in each group. *** P < 0·001.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The physiological importance of NO as a modulator of cellular function is now widely recognized and previous histochemical and immunocytochemical evidence has demonstrated the occurrence of a constitutive isoform of NO synthase (cNOS) in the endocrine pancreas (Panagiotidis et al. 1992a, 1994a; Schmidt et al. 1992; Corbett et al. 1993). This was recently verified by direct biochemical determination of islet cNOS activity (Salehi et al. 1996). Thus, the islet cNOS was found to be dependent on both Ca²⁺ and calmodulin and was suppressed by the NOS inhibitor L-NAME. This effect of L-NAME was enantiomerically specific (Salehi et al. 1996).

Insulin secretion

The functional importance of cNOS activity for insulin releasing mechanisms is not clear. Thus it was originally proposed (Laychock et al. 1991; Schmidt et al. 1992) that NO was a positive modulator of insulin release stimulated by glucose and L-arginine. However, previous in vitro and in vivo studies in our laboratory suggested rather that NO is a negative modulator of insulin secretion induced by both L-arginine and glucose (Panagiotidis et al. 1992a, 1994a, 1995; Salehi et al. 1996; Åkesson et al. 1996; Henningsson & Lundquist, 1998; Salehi et al. 1998). In fact the most recent studies from other laboratories also favour NO being inhibitory to insulin release stimulated by these secretagogues (Cunningham et al. 1994; Gross et al. 1995; Antoine et al. 1996).

In accordance with our previous data from experiments with glucose and L-arginine as secretagogues the present investigation also shows that cholinergic muscarinic stimulation of insulin release by carbachol is inhibited by NO production. It is known from studies on endothelial cells (Moncada et al. 1991) that cholinergic receptor activation leads to an increase in cytosolic calcium and an increased metabolism of L-arginine, which activates cNOS to give off a short burst of NO. A similar chain of events is also likely to occur in the islets of Langerhans, which for long have been known to be richly innervated by cholinergic nerves and furthermore both - and -cells are equipped with muscarinic cholinergic receptors and secrete insulin and glucagon, respectively, in response to cholinergic stimulation both during physiological and pharmacological conditions (Woods & Porte, 1974; Miller, 1981). In fact, cholinergic stimulation and modulation of the phospholipase C system is considered of utmost importance for a proper, physiological regulation of insulin release (Zawalich & Rasmussen, 1990).

In the presence of the NOS inhibitor L-NAME a marked potentiation of cholinergically stimulated insulin release was evident within a wide range of carbachol concentrations (cf. Fig. 1) lowering the threshold for carbachol stimulation and increasing the maximal response. The different types of NOS inhibitors, i.e.the L-arginine analogues L-NAME and L-NMMA as well as the indazole compound 7-nitroindazole all greatly enhanced carbachol-stimulated insulin release, whereas the intracellular NO donor hydroxylamine induced a marked and dose-dependent suppression of the secretory process. L-NMMA, L-NAME and 7-nitroindazole potentiated carbachol-induced insulin release at concentrations of 1-5 mM but not at lower concentrations. Obviously millimolar concentrations of L-NAME, L-NMMA and 7-nitroindazole are required to achieve a significant suppression of cNOS activity in the islet -cells. This might be explained by the fact that L-NMMA, in addition to its NOS inhibitory property, reportedly is capable of a small but significant NOS-dependent generation of NO and that esterase activity (demethylation of L-NAME to N^G-nitro-L-arginine) is needed for L-NAME to fully exhibit its inhibitory action (Southan & Szabó, 1996). The NOS inhibitory action of 7-nitroindazole is known to involve multiple mechanisms and appears to affect both the pteridine and arginine binding sites of NOS (Southan & Szabó, 1996).

We have previously shown (Panagiotidis et al. 1995; Henningsson & Lundquist, 1998) that 5 mM L-NAME and 5 mM L-NMMA did not influence basal insulin release at 7 mM glucose. However at high concentrations of L-NAME (>5 mM) the action of the NOS inhibitor must be considered mainly non-specific since, at for example a 10 mM concentration, both L-NAME and its enantiomer D-NAME, which is devoid of NOS inhibitory properties (Knowles & Moncada, 1994), did potentiate, quantitatively and markedly, the carbachol-induced insulin release in a similar way. This is in agreement with our previous finding that both 10 mM L-NAME and 10 mM D-NAME markedly potentiated basal insulin release at 7 mM glucose (Panagiotidis et al. 1995). Moreover, it was recently shown (Smith et al. 1997; Weinhaus et al. 1997) that 10 mM concentrations (or higher) of both L-NAME (blocking of K_ATP channels) and L-NMMA (electrogenic depolarization) were able to increase [Ca²⁺]_i to a similar level as an equivalent concentration of L-arginine. However, in this context it should be taken into account that insulin release stimulated by L-arginine, but not by L-NAME or L-NMMA, is always partially inhibited because it is counteracted by the accompanying and substantial NO production induced by L-arginine itself (Henningsson & Lundquist, 1998). In the present study we found that 2·5 mM L-NAME displayed a specific NOS inhibitory property by greatly potentiating insulin release, whereas 2·5 mM D-NAME had no significant effect. However, a tendency to a D-NAME effect was noted. This was probably due to a slight side-effect by the drug on K_ATP channels (Krippeit-Drews et al. 1996). The same pattern was previously observed with regard to L-arginine-induced insulin release, which was greatly potentiated by 5 mM L-NAME but unaffected by 5 mM D-NAME (Panagiotidis et al. 1995). Hence, 5 mM L-NAME seems to yield a close to 'optimal' inhibition of NOS in isolated islets with only minor non-selective side-effects. Such an assumption is further supported by our observation that the major part of the potentiating effect of L-NAME on carbachol-stimulated insulin release was still evident in diazoxide-treated K⁺-depolarized islets. Moreover, we have recently shown, for the first time (B. Åkesson, R. Henningsson, A. Salehi & I. Lundquist, unpublished results), that 5 mM L-NAME as well as 5 mM L-NMMA suppressed islet cNOS activity by approximately 60 % concomitant with a marked potentiation of glucose-stimulated insulin release, whereas 500 µM concentrations of the two NOS inhibitors had no effect on either cNOS or insulin release. These data are thus in accordance with our present results on the effects of different concentrations of L-NAME and L-NMMA on carbachol-induced insulin release.

As mentioned above, further evidence for the action of L-NAME as a true NOS inhibitor can be inferred from our present data showing that, in the presence of the K_ATP channel opener diazoxide and after depolarization by K⁺, the potentiating effect of 5 mM L-NAME on carbachol-induced insulin release was still markedly significant. Hence, we interpret this L-NAME effect as being due mainly to a true NOS inhibitory action although a minor effect on the K_ATP channels cannot be ruled out. This is also evident from the experiments where we directly compared the effect of L-NMMA and L-NAME at 2·5 mM concentrations and found that the potentiating effect of L-NMMA on carbachol-induced insulin release was significantly lower than that of L-NAME. However, both NOS inhibitors induced a quantitatively similar potentiation of carbachol-stimulated insulin release at 1 mM suggesting no side-effect at all at this dose. Taken together the accumulated data suggest that NO is a strong negative modulator of cholinergic muscarinic insulin release probably acting through S-nitrosylation of regulatory proteins in the secretory process. Thus we have previously proposed (Lundquist et al. 1991; Panagiotidis et al. 1992a, 1994a,b, 1995) that the inhibitory action of free radicals such as NO and H₂O₂ on insulin secretory mechanisms might be exerted through interaction with certain thiol groups essential for insulin release. Oxidation of thiol groups (cf. Hellman et al. 1974; Ammon & Mark, 1985) and/or formation of S-nitrosothiols inducing an impairment of the balance of the -cell glutathione system may constitute important mechanisms for the inhibitory action of NO and H₂O₂ (Panagiotidis et al. 1992a, 1994a,b, 1995; Åkesson et al. 1996). In contrast, the stimulatory action of NO on glucagon release apparently is independent of such modifications of these essential thiol groups, since the NO-donor hydroxylamine did not inhibit but increased the release of glucagon (Panagiotidis et al. 1994a; Salehi et al. 1996; Henningsson & Lundquist, 1998; Salehi et al. 1998).

Glucagon secretion

With regard to the influence of NO on glucagon secreting mechanisms our present results indicate that the action of NO on cholinergic glucagon release is quite the opposite to its effect on insulin release. Inhibition of islet NOS by L-NAME, L-NMMA and 7-nitroindazole suppressed carbachol-induced glucagon release in isolated islets. The inhibitory effect by L-NAME on glucagon release seemed to be exerted largely independently of membrane depolarization events, since it was still evident in K⁺-depolarized islets. Interestingly L-NMMA and 7-nitroindazole displayed their inhibitory action on carbachol-induced glucagon release at lower concentrations than L-NAME. In fact, the effect of 2·5 mM L-NAME was of doubtful significance, whereas a 5 mM concentration of the drug inhibited glucagon release in both non-depolarized and depolarized islets. It is not inconceivable that this discrepancy might be due to the fact, as previously mentioned, that L-NMMA is known to directly inhibit NOS activity, whereas L-NAME has to be metabolized by an esterase to yield the active inhibitor, N^G-nitro-L-arginine. Reportedly (Southan & Szabó, 1996), the rate of this reaction may vary between different tissues and cell types. 7-Nitroindazole is known to display a differential uptake into different cell types (Southan & Szabó, 1996) and might be taken up more easily by the glucagon cells than by the insulin-producing -cells and thus be able to exert its NOS inhibitory effect in the glucagon cells at lower medium concentrations of the drug. The finding that millimolar concentrations of L-NMMA no longer inhibited carbachol-induced glucagon release is probably explained by a certain depolarizing action of L-NMMA at these concentrations (Smith et al. 1997; Weinhaus et al. 1997) thus counteracting its NOS inhibitory effect.

Addition of the intracellular NO donor hydroxylamine at a low concentration (3 µM) potentiated carbachol-stimulated glucagon release. Higher concentrations of hydroxylamine, however, did not influence glucagon release. This is in accordance with previous data showing that low concentrations of hydroxylamine (3 and 30 µM) could stimulate glucagon release in the presence of high glucose (16·7 mM) or high glucose + L-arginine, whereas greater concentrations of hydroxylamine (0·3 and 3 mM) were without effect (Panagiotidis et al. 1994a; Salehi et al. 1996). Further, hydroxylamine dose-dependently reversed the suppression of glucagon release induced by -ketoisocaproic acid (KIC) at a lower concentration range than was required for its inhibitory action on KIC-induced insulin release (Salehi et al. 1998). Hence, the glucagon cell seems to be more sensitive to NO (reacts to lower concentrations of hydroxylamine) than the insulin-producing -cell. In this context it should be noted that hydroxylamine exerted a dose-dependent suppression of cholinergically induced insulin release. This discrepancy suggests different or partly different mechanisms of action for NO in insulin and glucagon secretory processes. The detailed mechanism of action by which NO stimulates glucagon secretion is unclear. One of the major effects of NO is to activate guanylate cyclase and thereby increase cyclic GMP in certain target cells (Moncada et al. 1991). However, we have previously shown that incubation of islets with 300 µM hydroxylamine induced a 14-fold increase in islet cyclic GMP levels, whereas 3 µM hydroxylamine apparently was without effect (Panagiotidis et al. 1995). Since 300 µM hydroxylamine did not affect glucagon release it seems less likely that the cyclic GMP system is the main signalling effector. However, there is a possibility that low concentrations of hydroxylamine, in fact, increase the cyclic GMP levels in the glucagon cells, although this is not detected in whole islets because the glucagon cells comprise only 15-20 % of the islet cell population. Hence, it is not inconceivable that, similar to the NO effects in the -cell, high concentrations of hydroxylamine-derived NO can induce influences on the transduction mechanisms for glucagon release, which are not directly related to the cyclic GMP-system and thus may mask and even antagonize the cyclic GMP effect. In this context it is worth noting that very recent data (Henningsson et al. 1997) have revealed that carbon monoxide (CO) is produced within the islets of Langerhans. CO was found to be a positive modulator of glucagon release. A negative interaction between CO and NO has been reported from other tissues (Ingi et al. 1996) and such interactions might have implications for the outcome of the present experiments. Some of these problems might possibly be resolved in the future by the use of purified glucagon cells. Further, although non-specific effects of hydroxylamine itself cannot be completely ruled out, they are probably exerted at concentrations of approximately one order of magnitude greater than used in the present study (Zeng & Weigel, 1995). After all, it cannot be excluded that the stimulating effect of NO on glucagon release in fact is mediated, at least partially, by the cyclic GMP system in the glucagon cells. As mentioned above a putative stimulating effect by NO on the cyclic GMP system in the -cell is probably overshadowed by the strong inhibitory action on insulin release exerted through S-nitrosylation of critical thiol groups.

In vivo effects on hormone secretion

Finally, our comparative in vivo data add favourably to our in vitro results and strongly suggest that NO in fact is a negative modulator of insulin secretion and a positive modulator of glucagon secretion. It is appreciated that the in vivo injection of L-NAME may induce multiple indirect effects on islet hormone secretion. Therefore, we studied the rapid initial peak of insulin and glucagon secretion and injected L-NAME at a very short time interval before carbachol administration. Similar in vivo effects were previously observed when L-NAME was administered together with L-arginine (Åkesson et al. 1996) or glucose (Salehi et al. 1996). On the other hand, insulin and glucagon release induced by injection of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) has been found to be unaffected by L-NAME (B. Åkesson & I. Lundquist, unpublished results) suggesting that L-NAME did not induce a non-specific potentiation of stimulated insulin release. Further, since the L-NAME-induced in vivo action brought about an increase in insulin release but a suppression of glucagon release it seems unlikely that effects of L-NAME on islet blood flow could have a significant influence on the results. Moreover, previous studies (Gross et al. 1995) did not reveal any specific effects of NOS inhibition on islet blood flow in the isolated perfused pancreas. The fact that the in vivo results were in perfect agreement with the data obtained with isolated islets argues in favour of the observed changes in plasma insulin and glucagon levels being mainly due to direct effects of L-NAME and carbachol on the pancreatic islets. Such an assumption has been verified very recently since we have found that islet cNOS activity is inhibited by approximately 55 % at 2 min following an I.V. injection of L-NAME at a dose which greatly enhanced the in vivo glucose-induced insulin response and suppressed the plasma glucagon levels (B. Åkesson, R. Henningsson, A. Salehi & I. Lundquist, unpublished results).

Conclusion

In conclusion, in the present paper we have demonstrated that the islet NO system is a negative modulator of insulin release and a positive modulator of glucagon release induced by cholinergic muscarinic stimulation. This could be shown both in vitro and in vivo by the use of the NOS inhibitors L-NAME, L-NMMA and 7-nitroindazole. Moreover, the intracellular NO donor hydroxylamine inhibited insulin release and increased glucagon release stimulated by carbachol. The action of NO on carbachol-induced islet hormone secretion was at least partially independent of membrane depolarization events, since the effects were still markedly evident in islets depolarized by high K⁺. The detailed elucidation of the regulatory influence of the islet NO system on insulin and glucagon secretion may have important implications for the physiology and pathophysiology of islet hormone secretion and hence for the development of diabetes mellitus.

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Acknowledgements

This study was supported by the Swedish Medical Research Council (14X-4286), the Swedish Diabetes Association, the Swedish Society of Medical Research, the Albert Påhlsson Foundation and the Åke Wiberg Foundation. The technical assistance of Elsy Ling and Britt-Marie Nilsson and the secretarial help of Eva Björkbom are gratefully acknowledged.

Corresponding author

I. Lundquist: Department of Pharmacology, Sölvegatan 10, S-223 62 Lund, Sweden.

Email: Ingmar.Lundquist{at}farm.lu.se

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