type 1 diabetes mellitus ( t1 dm ) is characterized by a loss of beta cell mass due to an autoimmune process . in a phase prior to the onset , immune cells infiltrate the islets creating an inflammatory microenvironment responsible for the -cell - specific toxicity . proinflammatory cytokines , such as interleukin-1 beta ( il-1 ) , tumor necrosis factor alpha ( tnf- ) , and interferon gamma ( ifn- ) , secreted by activated lymphocytes and macrophages during insulitis , induce nfkb mediated inos expression which has a key role in -cell apoptosis in the early t1 dm stage . it is produced in tissues by the enzyme nitric oxide synthase ( nos ) by oxidation of the amino acid l - arginine . there are three different tissue - specific nos isoforms : ca / calmodulin - independent and inducible nos ( inos ) , neuronal nos ( nnos ) , and endothelial nos ( enos ) . these last two isoforms also exhibit constitutive expression and ca / calmodulin - dependent activity . expression of nos isoforms has been studied in pancreatic islets , and the nnos form has been observed in -cell cells , located , mainly , in insulin secretory granules . it exhibits cytochrome c reductase activity in addition to no production activity , and a balance between both functions is essential for a normal insulin secretion in response to glucose stimulation . in contrast , enos expression has not been found in rat islets under basal conditions [ 2 , 3 ] . prooxidant agents formed from no induce damages in dna which activate p53 which induces proapoptotic expression of genes such as bax , fas , noxa , and puma . a p53-independent mechanism has been described in which no acts by inhibiting the sarcoendoplasmic reticulum pump ca atpase 2b ( serca2b ) protein and , subsequently , by inducing the endoplasmic reticulum stress mechanisms [ 4 , 5 ] . the importance of the mitochondrial pathway has also been highlighted in no - mediated -cell cell apoptosis [ 6 , 7 ] . paradoxically , an antiapoptotic effect of low concentration of no has been reported in several systems , including  cells [ 812 ] . in addition to apoptosis , cell proliferation has been described as a no - regulated process . although certain activating effects have been reported in physiological systems [ 13 , 14 ] , the main role of no in cellular proliferation is inhibitory . in the subventricular zone , no induces inhibition of stem cell proliferation by a nitrosylation process . other proposed mechanism for no antiproliferative action is a g1-s inhibition mediated by an induction of the cell cycle inhibitor p21   or cyclins inhibition . recently , no - mediated neogenesis stimulation has been observed in an alloxan - induced murine model of diabetes . a proinflammatory cytokine - mediated inhibition of cultured -cell proliferation in addition to the apoptotic effect has been observed by our group . as described previously , no production is one of the most important events mediated by proinflammatory cytokines . therefore , the aim of this study was to assess the importance of no in the cytokine - mediated antiproliferative effect on cultured  cells and to determine the role(s ) of different no synthase isoforms present in pancreatic islets . all animal procedures were performed with the approval of the cdiz university school of medicine ( cdiz , spain ) committee for the ethical use and care of experimental animals . bio - breeding ( bb ) and wistar rats were kept under conventional conditions in an environment - controlled room ( 20 - 21c , 12  h light - dark cycle ) with water and standard laboratory rat chow available ad libitum and their weight was daily recovered . blood extracted from the tail vein was used in bb rats for weekly random glucose measurements using an automatic glucose monitor ( accu - chek optimum , roche diagnostic , basel , switzerland ) . pancreatic islets were isolated from adult male wistar rats as described previously by mcdaniel et al . . louis , mo , usa ) supplemented with 2  mm l - glutamine ( gibco invitrogen , carlsbad , ca , usa ) , 100  u / ml penicillin , 100  g / ml streptomycin ( pen - strep , bio - whittaker europe , verviers , belgium ) , and 10% fetal bovine serum ( fbs , gibco invitrogen , carlsbad , ca , usa ) . proinflammatory cytokines ( peprotech ec ltd , london , uk ) used in the experiments were recombinant human il-1 ( 50  u / ml ) , recombinant rat ifn- ( 1000  u / ml ) , and recombinant rat tnf- ( 1000  u / ml ) . these concentrations were selected as being appropriate based on the results of previous published studies [ 20 , 21 ] . no donors , s - nitroso - n - acetyl - dl - penicillamine ( snap ) , and diethylenetriamine / nitric oxide adduct ( deta - no ) , obtained from sigma - aldrich ( st . louis , mo , usa ) show different no release rates . deta - no is a member of the nonoates family and has a half - life ( t1/2 ) of 20  h at 37c , ph 7.4 . the t1/2 of snap is approximately 6  h at 37c and ph 68 although its biological activity is highly influenced by the molecular environment of the parent thiol . inhibitors of nos 7-nitroindazole ( 7-ni ) , n-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide , dihydrochloride ( 1400  w ) , and n5-(1-iminoethyl)-l - ornithine , dihydrochloride ( l - nio ) , purchased from sigma - aldrich ( st . louis , mo , usa ) were used to inhibit the activity of neuronal , inducible , and endothelial isoforms of nos , respectively . proliferating  cells were detected using 5-bromo-2-deoxyuridine ( brdu ) 5  mol / l label ( sigma - aldrich , st . louis , mo , usa ) which was added to the cultures from the start of the assay together with the drugs being tested , when appropriate . following the appropriate culture duration , the islets were recovered and incubated for 15  min with trypsin - edta ( 0.25% trypsin , 1  mm edta ) in hanks ' balanced salt solution without ca and mg ( gibco invitrogen , carlsbad , ca , usa ) at 37c , and the islets were gently dispersed . after washing with pbs , cells were cytospin on poly - l - lysine - coated slides and fixed in 4% methanol - free formaldehyde . slides were immuno - stained using monoclonal mouse anti - brdu ( dako cytomation , denmark ) and polyclonal guinea pig anti - insulin ( sigma - aldrich , st . cells were permeabilized by incubation for 30  min with 0.1% triton - x100 in pbs and washed twice with 100  mm glycine buffer containing 0.1% triton - x100 and 3% bovine serum albumin . cells were then treated with hcl ( 2n ) in pbs for 30  min , neutralized with borax / borate buffer ( 0.1  m , ph 8.9 ) for 30  min , washed , and incubated overnight at 4c with anti - brdu and anti - insulin antibodies . stained cells were revealed using anti - mouse igg antibody ( alexa-546 conjugated ) and anti - guinea pig igg ( alexa-488 conjugated ) antibody ( molecular probes inc , eugene , or , usa ) . cell nuclei were stained with 4-6-diamidino-2-phenylindole ( dapi ) . to determine the proliferating fraction , total and insulin - positive / brdu - positive cells were analyzed using a fluorescence microscope in randomized conditions , by a single investigator . for statistical purposes , 48  h cultured islets in presence and absence of cytokines were processed for histology study by histogel and paraffin inclusion . 10  m sections were stained using a polyclonal rabbit anti - inos ( ab15323 , abcam , cambridge , uk ) and a polyclonal guinea pig anti - insulin ( sigma - aldrich , st . louis , mo ) and examined using a microscope equipped with a digital camera and the image analysis celldsoftware ( olympus , hamburg , germany ) . randomly grouped bb rats daily received an intraperitoneal injection of vehicle ( 0.1% dmso diluted in injection water ) alone or containing l - nmma at a dose of 30  mg / kg , from 4 to 9 weeks of age . animals were sacrificed at 4 ( before treatment ) , 7 , and 9 weeks of age . wistar , bb , and thymectomized bb rats were fasted overnight ( 1618  h ) and a blood sample was collected from the tail vein ( fasting or 0  min sample ) . then , an intraperitoneal injection of 40% solution of glucose was administered ( 2  g / kg ) , followed by blood sampling at 15 , 30 , 60 , and 120  min after the glucose administration . glycemia was measured with an automatic glucose monitor ( accu - chek optimum , roche diagnostic , basel , switzerland ) . blood samples obtained from fasting and 15  min after glucose administration were also used to plasma extraction and insulin quantification by elisa technique ( insulin rat ultrasensitive kit . weeks of age bb rat pancreas were resected , weighed , fixed in bouin 's solution for 6  h , and postfixed in formalin for 12  h. then , they were dehydrated , paraffin embedded , and longitudinal 10  m microtome sections were obtained . insulin was stained in obtained pancreas by immunohistochemical techniques using a mouse anti - rat insulin monoclonal antibody and a peroxidase conjugated goat anti - mouse igg antibody and revealed with dab kit ( sigma aldrich , st . insulin - positive areas were quantified in two complete sections of each pancreas using a microscope equipped with a digital camera and the image analysis cell d software ( olympus , hamburg , germany ) . -cell mass values were calculated by multiplying the total insulin - positive area / total pancreatic area ratio by the total pancreas weight . equivalent numbers of islets under the different experimental conditions described previously were lysed in lysis buffer ( 125  mm tris - hcl buffer ph 68 , 2% sds , 1  mm dtt and containing protease and phosphatase inhibitors ) . protein ( 40  g ) was loaded and electrophoresed on 8% sds - page . proteins were transfer - blotted on to polyvinylidene fluoride ( pdfv ) membranes and then incubated in blocking buffer ( 5% nonfat milk in 10  mm tris - hcl , 1.15  m nacl and 0.1% tween-20 ) for 1  h at room temperature . the blots were then incubated with rabbit polyclonal antibody against enos ( santa cruz biotechnology , santa cruz ca , usa ) and inos ( transduction laboratories bd , nj , usa ) overnight according to the manufacturer 's instructions , followed by incubation with peroxidase conjugated anti - rabbit igg for 1  h at room temperature . stained bands were revealed using immun - star western c kit ( bio - rad , hercules ca , usa ) and quantified by quantity one software version upgrade ( bio - rad , hercules , ca , usa ) . -tubulin expression was used as loading control and enos expression was calculated as ratio of enos : -tubulin densities . histological examination of 7 and 9 weeks of age rat pancreatic islets was performed in harris ' h&e stained pancreas sections using 20 objective lens . the severity of insulitis was graded as a function of the mononuclear cell infiltration of the pancreatic islets : 0 = no infiltrate ; 1 = periductular infiltrate ; 2 = periislet infiltrate ; 3 = intraislet infiltrate ; 4 = intraislet infiltrate associated with -beta cell destruction . thirty islets were examined in each pancreas and the mean score was calculated by dividing the total score by the number of islets examined . results are presented as means  sem of measurements performed in at least 3 animals . to determine the role of no donors on -cell proliferation , pancreatic islets were treated with proinflammatory cytokines or different concentrations of no donors snap and deta , alone or in combination with the caspase-3 inhibitor z - vad - fmk . -cell proliferation measured by brdu incorporation showed that the no donors , deta - no ( figure 1(a ) ) and snap ( figure 1(b ) ) exert an antiproliferative action on  cells in a dose - dependent manner . this effect of no donors was not modified by addition of z - vad - fmk to the cultures . to determine the contribution of no to the antiproliferative effect of proinflammatory cytokines on pancreatic  cells , pancreatic islets were cultured over a 48  h period and treated with proinflammatory cytokines , alone or in the presence of l - nmma ( an inhibitor of nitric oxide synthase ) . inhibition of -cell proliferation induced by proinflammatory cytokines was completely abolished by l - nmma treatment ( figure 2 ) . guanylate cyclase is the enzyme through which no exerts the greater part of its effect . to assess the implication of the guanylate cyclase pathway in the no effect on -cell proliferation , cultures of pancreatic islet were treated for 48  h with 8br - cgmp ( a guanylate cyclase analogue ) and its inhibitor odq , alone or in combination with proinflammatory cytokines . as shown in figure 3 , the cgmp analogue exerts no effect on -cell proliferation , and the antiproliferative effect of cytokines is not altered by the cgmp inhibitor odq .   to study the role of constitutive and inducible nos isoforms in the antiproliferative action of cytokines , cultured pancreatic islets were treated with proinflammatory cytokines in addition to 1400  w and 7-ni , inhibitors of inducible and constitutive isoforms of nitric oxide synthase , respectively ; the resultant -cell proliferation was quantified . the antiproliferative effect of proinflammatory cytokines underwent no modification when islet cultures were incubated with the constitutive nos inhibitor ( figure 4(e ) ) . related to inos , cytokine - dependent induction of protein was observed from 24  h of culture ( figure 4(a ) ) . inos expression study by immunohistochemical techniques in control cultured pancreatic islets showed a scarce stain with peripheral profile . however , cultured islet in presence of cytokines showed an increased inos expression localized internally in the islet ( figure 4(b ) ) . the specific inhibitor 1400  w partially reverted cytokine - induced decreased proliferation in a dose - dependent manner ( figure 4(d ) ) . endothelial isoform of nos expression was initially examined in pancreatic islets cultured under control condition , or after stimulation with proinflammatory cytokines . as shown in figure 4(c ) , enos isoform was expressed in pancreatic islets and was not altered by exposure to proinflammatory cytokines . to study the role of enos on the antiproliferative effect of cytokines , l - nio ( an inhibitor with higher affinity for enos than the other isoforms ) a partial inhibition of the antiproliferative effect of proinflammatory cytokines was observed in treated cultures ( figure 4(f ) ) mediated by l - nio . weight and random glycemia monitoring along treatment period show no differences in those parameters between control and l - nmma - treated animals . glucose homeostasis was studied by ipgtt and insulin determination before and 15  min after intraperitoneal glucose administration . as shown in figure 5 , ipgtt displays a normal curve at studied times in l - nmma treated as in control animals ( figure 6(a ) ) . in addition , insulin quantification also shows no changes between control and treated animals at none of studied times ( figure 6(b ) ) . bb rats treated with l - nmma from 4 to 7 or 9 weeks of age were sacrificed and pancreas was removed from each rat to quantify beta cell mass by immunohistological techniques . in figure 7 , a beta cell mass loss undergone by bb rats between 7 and 9 weeks of age can be observed . l - nmma reverts this effect at both studied times . to test the effect of l - nmma treatment on infiltration levels , h&e - stained sections obtained from treated and control bb rats were evaluated and infiltration scores were calculated . no changes were observed in infiltration level in response to l - nmma at none of studied times ( figure 8) . currently , no is considered an important mediator of cell signaling via its classical guanylate cyclase pathway or by exerting posttranslational modifications on an increasing number of proteins and hence regulating their activity . in the present study , we analyzed the role of no in the previously described antiproliferative action of proinflammatory cytokines on pancreatic  cells . loss of membrane integrity and cytochrome c release induced by deta - no and snap in insulin producing cell lines has been reported [ 22 , 23 ] . in our system , no donors snap and deta - no induced a decrease in cultured -cell proliferation at concentrations up to 50  m for deta - no and up to 400  m for snap . this effect is dose dependent and similar to that obtained with proinflammatory cytokines ( il-1  +  tnf-  +  ifn- ) treatment ( figure 1 ) . the antiproliferative effect observed in response to no donors is not dependent on the biological t1/2 and suggests that no exerts its biological action in the first hours after exposure . to discard the possibility that the decrease in proliferation was due to a cytokine - induced apoptosis in proliferating  cell , we treated pancreatic -cell cultures with the caspase-3 inhibitor zvadfmk , in addition to the no donors . -cell apoptosis inhibition induced by zvadfmk exerts no effect on the antiproliferative effect of no donors suggesting that the two processes are unrelated . although studies in neonatal rat islets indicated that cytokine - induced no production regulates the expression of up to 42 proteins , a considerable number of proteins are regulated in a no - independent manner . thus , the cytokine - induced apoptosis in mouse  cells is not caused by the inducible isoform of nos alone . endoplasmic reticulum stress has been described as a possible mechanism by which no induces apoptosis . its presence regulates unfolded protein response ( upr ) signaling pathway that determines apoptotic response to reticulum stress . in addition to cytokines , there is evidence of no participation in mitochondrial dysfunction and cell death occurring in pc12 cells in response to amyloid  . results from our study suggest that , in addition to apoptosis , no can be implicated in the antiproliferative effect of proinflammatory cytokines on pancreatic  cell since treatment with nos inhibitor l - nmma is capable of reverting this cytokine - induced effect ( figure 2 ) . very few studies have investigated the possible mechanisms underlying the effect of no on cellular proliferation . in pulmonary microvascular smooth muscle cells , exogenous no upregulates p21 , a protein which mediates cell cycle arrest . further , no is capable of inhibiting erk 1/2 and akt activation in breast cancer cell lines and pancreatic  cells [ 29 , 30 ] . these results are in accord with those observed by our group ; that is , proinflammatory cytokines induced an inhibition of erk 1/2 in islet cultures   and in early stages of insulitis . however , no has also been described as an inducer of proliferation , when acting at low concentrations . for example , in mesangial cells , the no donor snap increases proliferation at concentrations of up to 200  m and inhibits proliferation at higher concentrations , in a process implying akt signaling and cox-2 protein . activation of no - sensitive guanylyl cyclase by no binding to the enzyme 's prosthetic heme group has been described as one of the most important no - mediated cellular effects . nevertheless , results observed in our system suggest that this pathway is not involved in the no - mediated antiproliferative effect observed in  cells from islets treated with proinflammatory cytokines ( figure 4 ) . these results are in accord with those previously reported in tumor cell lines in which the proliferative effect of low no concentration is mediated by the cgmp pathway , but the antiproliferative effect induced by high concentrations of no is independent of this pathway . a proposed cgmp - independent mechanism of no action is its direct interaction with protein and , as such , performing posttranslational modifications such as nitration or s - nitrosylation . for example , s - nitrosylation of egf receptor has been reported in human neuroblastoma nb69 as a mechanism of no - mediated antiproliferative effect . the role of nos isoforms in no production in response to proinflammatory cytokines has not been well defined . our results show that inos , the expression of which is induced in our system after 24  h culture in the presence of proinflammatory cytokines , is implicated while constitutive isoform is not implicated in cytokine - induced antiproliferative response in  cells ( figure 4 ) . of note is that although inos inhibitor 1400  w inhibited the cytokine - mediated antiproliferative effect , its effect was only partial , and was unlike the total reversion by l - nmma ( a general nos inhibitor ) . it is also interesting the effect of 1400  w increasing beta cell proliferation in absence of cytokines which could be due to a certain level of islet cells inos activation induced by islet isolation process . this partial effect of 1400  w suggests that other nos isoforms are participating in antiproliferative response to cytokines . endothelial nos determination by western blotting was performed in cultured rat islets in the presence of proinflammatory cytokines and , contrary to the results reported by lajoix et al . , enos expression was observed . this expression is maintained in culture and was not affected by the presence of cytokines ( figure 4(b ) ) and could be attributed to endothelial cells in the profuse vascular network present in pancreatic islets . once the presence of enos in islets is confirmed , its implication in -cell antiproliferative response to cytokines was tested in islet cultures treated with l - nio   ( an inhibitor whose primary target is enos ) alone or in combination with cytokines . l - nio induced a partial reversion of cytokine - induced -cell antiproliferation in a similar manner to that induced by the inos inhibitor 1400  w. these results suggest participation of both no isoforms in the antiproliferative response induced by proinflammatory cytokines , possibly by enos producing no in a paracrine manner . in this sense , it has recently reported that inflammatory environment induces changes in enos which may adopt functional features of inos in a ca - independent way . to assess no involvement in the pathophysiology of t1 dm , we treated biobreeding rats , an animal model of autoimmune diabetes , with the general nos inhibitor l - nmma at early stages of the insulitis process . previous observations from our group indicated a halting of -cell proliferation between 4 and 7 weeks of age and which can be reversed by administering neutralizing antibody against ifn- . treatment with l - nmma of bb rats from 5 weeks of age induced a reversal of -cell proliferation loss observed in untreated animals ( figure 5 ) . in addition , a relative recovery of -cell area with a regional pattern in treated animals at 7 weeks of age was observed . the effects , either on proliferation of  cell or on -cell area , were very much more evident in the head than in the tail of the pancreas . a similar regional pattern of response was reported in earlier studies using a malnutrition model . a plausible explanation for this regional effect could be a different vascularization in the head area compared to that in the tail which precluded equivalent drug availability in all areas of the pancreas volume . further , a different response to environmental factors due to a different islet composition could underlie this observed effect . t1 dm prevention strategies focus , essentially , on early stages of the disease before clinical onset when there is enough -cell mass in the pancreas to maintain its metabolic function . our present study highlights the importance of no as a mediator of the observed impaired -cell proliferative response from the initial stages of insulitis . this impairment in proliferative capacity precludes an appropriate regenerative response of  cells to autoimmune damage . our data also highlight the property of l - nmma to maintain -cell proliferative capacity in early insulitis stage and , as such , confirms that nitric oxide might be an important therapeutic target in the strategies of t1 dm prevention .