The effects of cold acclimation and nitric oxide on antioxidative enzymes in rat pancreas

Alterations of pancreatic antioxidative defense (AD) and possible nitric oxide (NO) role in AD organization of adult rats receiving l-arginine.HCl (2.25%) or N(omega)-nitro-l-arginine methyl ester (L-NAME.HCl, 0.01%) as drinking liquids and maintained at room (22+/-1 degrees C) or low (4+/-1 degrees C) temperature for 45 days were studied. For that purpose, copper, zinc- and manganese superoxide dismutase (CuZnSOD, MnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and glutathione reductase (GR) activities were determined. Cold-induced decrease of CuZnSOD was inhibited with L-NAME, while l-arginine produced the same effect as cold in both supplemented groups. Cold acclimation elevated GSH-Px activity. l-Arginine and L-NAME expressed no effect on GSH-Px in rats kept at room temperature. L-NAME additionally elevated cold-induced GSH-Px activity, l-arginine expressing a similar trend. Cold-induced increase in GST activity was inhibited by L-NAME, while l-arginine inhibited this enzyme in both supplemented groups. Cold acclimation increased GR activity in control and L-NAME-treated group and l-arginine expressed a similar trend. Neither of the treatments affected MnSOD and CAT activities. Cold-induced changes of pancreatic AD were additionally affected by the alterations in l-arginine-NO-producing pathway. Some AD changes in the same direction with l-arginine or L-NAME point to the complexity of nitrogen compounds metabolism and function, accompanied by tissue-specific response.     

Inducible nitric oxide synthase immunoreactivity in healthy rat pancreas

Nitric oxide (NO) is produced by NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). It is believed that, while nNOS and eNOS are effective in regulation of normal physiological processes, iNOS is expressed at an increasing rate especially in inflammatory process. The aim of this study was to determine the presence of iNOS immunoreactivity (iNOS-IR) and, to compare the iNOS-IR in islet of Langerhans cells (LC), acinar cells (AC), centroacinar cells (CC) and ductal cells (DC) by immunohistochemical (IHC) method in healthy rat pancreata. This study revealed the presence of iNOS-IR in all cell types except AC. Statistical analysis revealed a highly significant difference (p<0.001) with respect to iNOS-IR in comparison of all cell types. However, binary comparison of cell types revealed no significant differences between LC and DC (p=0.136), significant differences LC and CC, CC and DC (p=0.001 and 0.022, respectively) and a highly significant differences LC and AC, AC and DC (P<0.001). The results of this study indicate that iNOS-IR is present in almost all LC. Thus, especially in reseach related to diabetes, it should not be disregarded that iNOS may be constitutively present in pancreatic islets.     

Effect of nitric oxide on the somatostatinergic system in the rat exocrine pancreas

Nitric oxide (NO) and somatostatin (SS) are two important mediators of the exocrine and endocrine pancreas, exerting opposite effects on this organ. There is strong evidence suggesting an interaction between pancreatic NO and SS. The aim of this study was to determine whether L-arginine (L-Arg), the substrate for NO synthase (NOS), and Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, regulate pancreatic somatostatin-like immunoreactivity (SSLI) content and the SS mechanism of action in pancreatic acinar cell membranes. L-Arg (150 mg/kg, intraperitoneally (i.p.)), L-NAME (50 mg/kg, i.p.) or L-NAME plus L-Arg were injected twice daily at 8 h intervals for 8 days. L-Arg decreased pancreatic SSLI content as well as the number of SS receptors in pancreatic acinar cell membranes whereas L-NAME increased both parameters. The stable SS analogue SMS 201-995 induced a significantly lower inhibition of forskolin-stimulated adenylyl cyclase activity in pancreatic acinar cell membranes from L-Arg-treated rats whereas an increased inhibition was observed in pancreatic acinar membranes from L-NAME-treated rats. These results indicate that the NO system may contribute to the regulation of the pancreatic somatostatinergic system.     

Cholecystokinin octapeptide induces both proliferation and apoptosis in the rat pancreas

Cholecystokinin-8 (CCK-8) causes exocrine pancreatic hypertrophy and hyperplasia. High doses of the CCK analogue cerulein causes necrosis and an inflammatory response in the pancreas. We have studied the pancreatic growth response in rats after administration of CCK-8 for 3 days, given either intermittently (20-80 microg/kg) twice a day, or continuously (2.4-48 microg/kg per 24 h). Plasma CCK-8 levels, pancreatic wet weight, water, protein and DNA contents and the pancreatic caspase-3 activity were measured. Cell proliferation was visualized by [3H]thymidine incorporation and apoptosis by TUNEL reaction. Continuous administration of CCK-8 dose-dependently increased the plasma CCK levels, the pancreatic wet weight, protein and DNA contents as well as thymidine labeling index, apoptotic index and caspase-3 activity. Intermittent injections of CCK-8 caused transient raises in plasma CCK, increased apoptotic index and caspase-3 activity, a dose-dependent increase in thymidine labeling but caused a dose-dependent reduction of pancreatic wet weight, protein, and DNA contents. It is concluded that CCK-8 causes both increased proliferation and apoptosis in the pancreas. In case of continuous administration of CCK-8, the proliferation outweighs the apoptosis causing hyperplasia but in the case of intermittent administration the opposite effect is seen.     

Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation

In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NADPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas.     

Exogenous,basal, and flow-induced nitric oxide production and endothelial cell proliferation

The role of nitric oxide (NO) from endogenous and exogenous sources in regulating large vessel and microvascular endothelial cell proliferation was investigated. Exogenous NO liberated from five different chemical donors inhibited bovine aortic, bovine retinal microvascular, and human umbilical vein endothelial cell proliferation in a dose-dependent manner as determined by ³H-thymidine incorporation. The potency of the donors varied as a function of the donors' half-lives. Donors with half-lives greater than 30 min were more effective than donors with significantly shorter half-lives. Coincubation of endothelial cells with 0.4 mM deoxyadenosine and 0.4 mM deoxyguanosine reduced the percentage of inhibition due to an NO donor. These data are consistent with a ribonucleotide reductase-dependent mechanism of inhibition. Inhibition of basal NO production with four different inhibitors of nitric oxide synthase (NOS) did not modify proliferation. Laminar flow with a wall shear stress of 22 dyn/cm²inhibited the proliferation of subconfluent bovine aortic endothelial cells. The addition of a NOS inhibitor did not abrogate the flow-induced inhibition of proliferation, suggesting that flow-stimulated release of NO from endothelial cells did not account for flow-induced inhibition of proliferation. Taken together, these data suggest that relatively large concentrations of exogenous NO inhibit endothelial cell proliferation, while endogenous levels of NO are inadequate to inhibit proliferation. J. Cell. Physiol. 171:252–258, 1997. © 1997 Wiley-Liss, Inc.     

Immunohistochemical detection of apoptosis, proliferation and inducible nitric oxide synthase in rat urothelium damaged by cyclophosphamide treatment

The present study was conducted to investigate cell death, proliferation and inducible nitric oxide synthase (iNOS) immunoreactivity in rat urothelium within 24 h after a single intraperitoneal dose of cyclophosphamide (CP). Necrotic cells were identified predominantly in the superficial cell layer from 1 h until 6 h after CP injection, most of them desquamating from the urothelium into the lumen of the urinary bladder. Active caspase-3 immunohistochemistry revealed apoptotic cells from 12 h until 24 h after CP injection. The apoptotic index reached a peak at 18 h and then rapidly dropped. Simultaneously with the decrease of apoptosis, the proliferation index increased from 18 h until 24 h after CP treatment. Immunoreaction to iNOS was first detected at 6 h in basal and intermediate cells. Later, iNOS immunoreactivity became stronger and was present in all cell layers. Our results suggest that the destruction of rat urothelium during 24 h after CP administration is due not only to necrosis, but also to apoptosis. The first 6 h are characterised by necrotic changes and no iNOS immunoreactivity. Thereafter, apoptosis and iNOS immunoreactivity are observed within the damaged urothelium. At 24 h after CP injection, iNOS immunoreactivity is still present, but proliferation prevails over cell death, enabling the urothelium to start regeneration.     

The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney

The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.     

The role of nitric oxide in the spatial heterogeneity of basal microvascular blood flow in the rat diaphragm

The effects of N-nitro-L-arginine (L-NOARG) and N^G-monomethyl-L-arginine (L-NMMA) on the spatialdistribution of diaphragmatic microvascular blood flow were assessed in anesthetized, mechanically ventilated rats. Microvascular blood flow was measured after different periods at either a fixed site (Q_stat) or 25 different sites (Q_scan) using computer-aided laser-Doppler flowmetry (LDF) scanning. The value of Q_stat was unaffected after 15–20 min superfusion with any one of the following agents: L-NOARG (0.1 mM), L-NMMA (0.1 mM), L-arg (10 mM). The cumulative frequency histogram of the Q_scan value in the control group displayed a non-Gaussian distribution that was not significantly affected after 15 min superfusion with the vehicle of L-NOARG. Superfusion with either L-NMMA or L-NOARG at 0.1 mM for 15 min displaced the histogram of cumulative frequency to the left, with the median value of blood flow decreasing by 10 to 20%. However, skewness and kurtosis of the distribution of basal Q_scan were unaffected after superfusion of either of the L-arg analogues. Pretreatment with L-arg (10 mM), followed by co-administration of L-arg (10 mM) with L-NOARG (0.1 mM) only partially prevented L-NOARG from exerting this inhibitory effect on the distribution of basal Q_scan, while pretreatment with L-arg in the same manner could prevent L-NMMA from exerting its inhibitory effect. There was a weak but significant linear relationship between the magnitude of basal Q_scan and normalized changes in basal Q_scan after superfusion of either of the L-arg analogues. In conclusion, a basal NO activity is present in the diaphragmatic microvascular bed of rats. LDF scanning rather may yield more vivid information about the extent of overall tissue perfusion than conventional LDF whenever basal NO activity is involved. Moreover, the parallel flow profiles after NO synthase blockade suggest that the spatial inhomogeneity of basal diaphragmatic microvascular blood flow is not dependent on basal NO formation.     

Galanin inhibition of cholecystokinin-8-induced increase in [Ca2+]i in individual rat pancreatic B-cells.

The intracellular free Ca2+ ion concentration [( Ca2+]i) was measured in individual rat pancreatic B-cells loaded with fura-2. The cells were prepared by enzymatic digestion and fluorescence-activated cell sorting. The resting concentration of [Ca2+]i in B-cells was 126.3 +/- 3.1 nM in the presence of 4.4 mM glucose. Addition of cholecystokinin-8 (CCK-8) resulted in rapid and transient rises in [Ca2+]i. Perifusion of B-cells with galanin attenuated the amplitude and duration of CCK-8-induced [Ca2+]i changes and this inhibitory effect was concentration-dependent and reversible. Perifusion of B-cells with nifedipine, a voltage-sensitive Ca2+ channel blocker, reduced the duration of the [Ca2+]i increase induced by CCK-8, indicating that the Ca2+ entry from the extracellular space was, at least in part, involved in CCK-8-induced increases in [Ca2+]i.     

Pregnancy influence on the vascular interactions between nitric oxide and other endothelium-derived mediators in rat kidney

Peripheral vascular resistance and sensitivity to circulating pressor and vasoconstrictor agents are blunted during pregnancy. This has been mainly attributed to an increased production of endothelium-derived mediators. The objective of this work was to evaluate if pregnancy changes the relative participation of nitric oxide (NO) and prostaglandins (PG) in respect to the modulation of the increases in renal perfusion pressure induced by phenylephrine (Phe). Dose-response curves were made with gradually increasing doses of Phe using an isolated kidney preparation in the presence of a NO synthase (NOS) inhibitor (L-NAME, 1 microM), a PG-synthesis inhibitor (indomethacin, 1 microM), both, or neither. Also, renal cyclooxygenase (COX-1 and COX-2) and endothelial NOS (eNOS) expression was determined using PCR. The experiments were done in kidneys from nonpregnant and pregnant rats. Our results showed that the relative participation of renal vasoactive mediators seems to change during pregnancy. We found the presence of a COX-1-dependent vasoconstrictor in the middle of pregnancy that was not found in nonpregnant rats. Our results also suggest that there is increased participation of another renal vasodilator substance, the effect of which is observed when NO or PG synthesis is inhibited during late pregnancy. In addition, an apparent interaction between renal eNOS and COX-1 expression was observed: eNOS expression was diminished, while COX-1 was increased during the 2nd week of pregnancy. In contrast, in kidneys from the 3rd week of pregnancy, the expression of these two enzymes was similar.     

Cellular action of cholecystokinin-8S-mediated excitatory effects in the rat periaqueductal gray

The peptide cholecystokinin (CCK) is one of the major neurotransmitters modulating satiety, nociception, and anxiety behavior. Although many behavioral studies showing anti-analgesic and anxiogenic actions of CCK have been reported, less is known about its cellular action in the central nervous system (CNS). Therefore, we examined the action of CCK in rat dorsolateral periaqueductal gray (PAG) neurons using slice preparations and whole-cell patch-clamp recordings. Application of CCK-8S produced an inward current accompanied by increased spontaneous synaptic activities. The CCK-8S-induced inward current (I(CCK)) was recovered after washout and reproduced by multiple exposures. Current-voltage plots revealed that I(CCK) reversed near the equilibrium potential for K(+) ions with a decreased membrane conductance. When several K(+) channel blockers were used, application of CdCl(2), TEA, or apamin significantly reduced I(CCK). I(CCK) was also significantly reduced by the CCK(2) receptor antagonist, L-365,260, while it was not affected by the CCK(1) receptor antagonist, L-364,718. Furthermore, we examined the effects of CCK-8S on miniature excitatory postsynaptic currents (mEPSCs) in order to determine the mechanism of CCK-mediated increase on synaptic activities. We found that CCK-8S increased the frequency of mEPSCs, but had no effect on mEPSC amplitude. This presynaptic effect persisted in the presence of CdCl(2) or Ca(2+)-free bath solution, but was completely abolished by pre-treatment with BAPTA-AM, thapsigargin or L-365,260. Taken together, our results indicate that CCK can excite PAG neurons at both pre- and postsynaptic loci via the activation of CCK(2) receptors. These effects may be important for the effects of CCK on behavior and autonomic function that are mediated via PAG neurons.     

The localization of neuronal nitric oxide synthase may influence its role in neuronal precursor proliferation and synaptic maintenance

Neuronal nitric oxide synthase (nNOS) is implicated in some developmental processes, including neuronal survival, differentiation, and precursor proliferation. To define the roles of nNOS in neuronal development, we utilized the olfactory system as a model. We hypothesized that the role of nNOS may be influenced by its localization. nNOS expression was developmentally regulated in the olfactory system. During early postnatal development, nNOS was expressed in developing neurons in the olfactory epithelium (OE), while in the adult its expression was restricted to periglomerular (PG) cells in the olfactory bulb (OB). At postnatal week 1 (P1W), loss of nNOS due to targeted gene deletion resulted in a decrease in immature neurons in the OE due to decreased proliferation of neuronal precursors. While the pool of neuronal precursors and neurogenesis normalized in the nNOS null mouse by P6W, there was an overgrowth of mitral or tufted cells dendrites and a decreased number of active synapses in the OB. Cyclic GMP (cGMP) immunostaining was reduced in the OE and in the glomeruli of the OB at early postnatal and adult ages, respectively. Our results suggest that nNOS appears necessary for neurogenesis in the OE during early postnatal development and for glomerular organization in the OB in the adult. Thus, the location of nNOS, either within cell bodies or perisynaptically, may influence its developmental role.     

Characterization of basal nitric oxide production in living cells

Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.     

Effect of dalarginum on the level of nitric oxide stable metabolites in rat kidneys during apoptosis activation

The influence of dalarginum on the level of nitric oxide stable metabolites in the rat kidneys was investigated at various models of apoptosis activation (formed by two methods of acute renal failure). Dalarginum (synthetic leu-enkephaline) was entered in a dose of 100 micrograms/kg a day. Detection of apoptosis was carried out by determination of DNA fragmentation. Administration of dalarginum resulted in the significant decrease of NO--nitrite- and nitrate anions stable metabolites content, the latter is capable to be one of the mechanisms of the preparation antiapoptotic effect.     

The long-term effect of mesh bioprosthesis in inguinal hernia repair on testicular nitric oxide metabolism and apoptosis in rat testis

Polypropylene mesh is the most widely used material in inguinal hernia repair. Although polypropylene mesh is known as an inert material, it is experimentally proven that mesh generates a chronic inflammatory tissue reaction. The aim of the present study was to investigate the long-term effects of polypropylene mesh material used in inguinal hernia operations on testicular function, testicular nitric oxide (NO) metabolism and germ cell-specific apoptosis in rats. The study comprised 40 male rats that were randomly allocated into two groups. In group 1, the left spermatic cord was elevated and a 0.5 x 1 cm polypropylene mesh was placed behind the left inguinal spermatic cord and group 2 consisted of the sham-operated controls. Blood samples were taken at 6 months preoperatively and postoperatively after to assess luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels for hormonal evaluation. Testicular NO was evaluated by the Griess method, apoptosis by a TUNEL method and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expressions by immunohistochemical staining. Mild (+) eNOS expression was observed in all specimens. Mild (+) iNOS expression was only detected in ipsilateral testis of the mesh-implanted study group. Apoptotic cells were not detected in any samples. We are of the opinion that long-term polypropylene mesh implantation has no effect on testicular hormonal function and only a limited effect on nitric oxide levels and this effect is not sufficient to cause apoptosis in testis that could lead to infertility. It seems that mesh implantation is a reliable method in inguinal hernia repair; however, further work is required by more sensitive methods to fully elucidate the potential testicular damage.