Involvement of abscisic acid-dependent and -independent pathways in the upregulation of antioxidant enzyme activity during NaCl stress in cotton callus tissue

The role of abscisic acid (ABA) in the signal transduction pathway associated with NaCl-induced up-regulation of antioxidant enzyme activity was examined in a NaCl-tolerant cotton callus cell line treated with NaCl, ABA, paraquat, or H₂O₂ in the presence and absence or fluridone, an inhibitor of terpene, and therefore, ABA synthesis. Treatment with NaCl resulted in a rapid increase (within 30 minutes) in the ABA levels of the callus tissue, and the NaCl, ABA, and paraquat treatments induced rapid increases in the activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase. Pre-treatment with fluridone significantly suppressed the NaCl-induced increases, but only slightly delayed the increases in tissue subjected to exogenous ABA treatment. This implies that ABA is involved in the signal transduction pathway associated with the NaCl-induced up-regulation of these antioxidant enzymes. Pre-treatment with fluridone had no effect on the paraquat-induced increases, suggesting that these enzymes can also be up-regulated by a pathway other than the one mediated by ABA. Both the NaCl and paraquat treatments produced significant increases in the superoxide levels within the callus, but the increase resulting from the paraquat treatment was significantly higher than the increase resulting from the NaCl treatment. These data suggest that NaCl stress results in the production of reactive oxygen intermediates (ROI) which signals the induction of an ABA-dependent signaling pathway. The production of very high levels of ROI, such as those that occur with paraquat treatment or perhaps during periods of prolonged or extreme stress, may induce an ABA-independent signaling pathway.     

The effects of NaCl on antioxidant enzyme activities in callus tissue of salt-tolerant and salt-sensitive cotton cultivars (Gossypium hirsutum L.)

Summary To determine NaCl effects on callus growth and antioxidant activity, callus of a salt-tolerant and a salt-sensitive cultivar of cotton was grown on media amended with 0, 75, and 150 mM NaCl. Callus of the salt-tolerant cultivar, Acala 1517-8 8, grown at 150 mM NaCl, showed significant increases in superoxide dismutase, catalase, ascorbate peroxidase, peroxidase and glutathione reductase activities compared to callus tissue grown at 0 mM NaCl. In contrast, callus tissue of the salt-sensitive cultivar, Deltapine 50, grown at 0, 75, and 150 mM NaCl, showed no difference in the activities of these enzymes. At the 150 mM NaCl treatment, peroxidase was the only antioxidant enzyme from Deltapine 50 with an activity as high as that observed in Acala 1517-88. The NaCl-induced increase in the activity of these enzymes in Acala 1517-88 indicates that callus tissue from the more salt-tolerant cultivar has a higher capacity for scavenging and dismutating superoxide, an increased ability to decompose H₂O₂, and a more active ascorbate-glutathione cycle when grown on media amended with NaCl.     

Modulation of outer medullary NaCl transport and oxygenation by nitric oxide and superoxide

We expanded our region-based model of water and solute exchanges in the rat outer medulla to incorporate the transport of nitric oxide (NO) and superoxide (O(2)(-)) and to examine the impact of NO-O(2)(-) interactions on medullary thick ascending limb (mTAL) NaCl reabsorption and oxygen (O(2)) consumption, under both physiological and pathological conditions. Our results suggest that NaCl transport and the concentrating capacity of the outer medulla are substantially modulated by basal levels of NO and O(2)(-). Moreover, the effect of each solute on NaCl reabsorption cannot be considered in isolation, given the feedback loops resulting from three-way interactions between O(2), NO, and O(2)(-). Notwithstanding vasoactive effects, our model predicts that in the absence of O(2)(-)-mediated stimulation of NaCl active transport, the outer medullary concentrating capacity (evaluated as the collecting duct fluid osmolality at the outer-inner medullary junction) would be ～40% lower. Conversely, without NO-induced inhibition of NaCl active transport, the outer medullary concentrating capacity would increase by ～70%, but only if that anaerobic metabolism can provide up to half the maximal energy requirements of the outer medulla. The model suggests that in addition to scavenging NO, O(2)(-) modulates NO levels indirectly via its stimulation of mTAL metabolism, leading to reduction of O(2) as a substrate for NO. When O(2)(-) levels are raised 10-fold, as in hypertensive animals, mTAL NaCl reabsorption is significantly enhanced, even as the inefficient use of O(2) exacerbates hypoxia in the outer medulla. Conversely, an increase in tubular and vascular flows is predicted to substantially reduce mTAL NaCl reabsorption. In conclusion, our model suggests that the complex interactions between NO, O(2)(-), and O(2) significantly impact the O(2) balance and NaCl reabsorption in the outer medulla.     

Respective roles of nitric oxide and superoxide radical in the respiratory burst activity of rat polymorphonuclear leukocytes induced by hyperthyroidism

Summary

Administration of single doses of 0.1 mg of L-3,3′,5-triiodothyronine (T₃)/kg for 3 consecutive day to fed rats elicited a marked increase both in the opsonized zymosan-induced luminal-amplified integrated chemiluminescence (ICL) of isolated polymorphonuclear leukocytes (PMN) in the absence (200%) and presence (228%) of L-arginine, and in the rate of superoxide radical (O₂^.-) production (180%). In the presence of L-arginine, the ICL was significantly increased by 57 and 17% over values observed in its absence, in PMN from control rats and T₃-treated animals, respectively, an effect that was completely abolished by Nω-nitro-L-arginine. However, the net L-arginine-dependent ICL was comparable in stimulated PMN from both experimental groups, and the respective rates of nitric oxide (NO^.) production were not significantly different, either in the absence or presence of nitro-L-arginine methyl ester. It is concluded that thyroid hormone-induced respiratory burst activity of rat PMN is not dependent on changes in NO^. synthase activity, but rather on the adaptive increase in O₂^.- generation by NADPH oxidase.     

Inhibition of nitric oxide synthase enhances superoxide activity in canine kidney

To evaluate the role of a potential interaction between superoxide anion (O(2)(-)) and nitric oxide (NO) in regulating kidney function, we examined the renal responses to intra-arterial infusion of a superoxide dismutase mimetic, tempol (0.5 mg.kg(-1).min(-1)), in anesthetized dogs treated with or without NO synthase inhibitor, N(omega)-nitro-l-arginine (NLA; 50 microg.kg(-1).min(-1)). In one group of dogs (n = 10), tempol infusion alone for 30 min before NLA infusion did not cause any significant changes in renal blood flow (RBF; 5.2 +/- 0.4 to 5.0 +/- 0.4 ml.min(-1).g(-1)), glomerular filtration rate (GFR; 0.79 +/- 0.04 to 0.77 +/- 0.04 ml.min(-1).g(-1)), urine flow (V; 13.6 +/- 2.1 to 13.9 +/- 2.5 microl.min(-1).g(-1)), or sodium excretion (U(Na)V; 2.4 +/- 0.3 to 2.2 +/- 0.3 micromol.min(-1).g(-1)). Interestingly, when tempol was infused in another group of dogs (n = 12) pretreated with NLA, it caused increases in V (4.4 +/- 0.4 to 9.7 +/- 1.4 microl.min(-1).g(-1)) and in U(Na)V (0.7 +/- 0.1 to 1.3 +/- 0.2 micromol.min(-1).g(-1)) without affecting RBF or GFR. Although NO inhibition caused usual qualitative responses in both groups of dogs, the antidiuretic (47 +/- 5 vs. 26 +/- 4%) and antinatriuretic (67 +/- 4 vs. 45 +/- 11%) responses to NLA were seen much less in dogs pretreated with tempol. NLA infusion alone increased urinary excretion of 8-isoprostane (13.9 +/- 2.7 to 22.8 +/- 3.6 pg.min(-1).g(-1); n = 7), which returned to the control levels (11.6 +/- 3.4 pg.min(-1).g(-1)) during coadministration of tempol. These data suggest that NO synthase inhibition causes enhancement of endogenous O(2)(-) levels and support the hypothesis that NO plays a protective role against the actions of O(2)(-) in the kidney.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.     

Reactive nitroxidative species and nociceptive processing: determining the roles for nitric oxide, superoxide, and peroxynitrite in pain

Pain is a multidimensional perception and is modified at distinct regions of the neuroaxis. During enhanced pain, neuroplastic changes occur in the spinal and supraspinal nociceptive modulating centers and may result in a hypersensitive state termed central sensitization, which is thought to contribute to chronic pain states. Central sensitization culminates in hyperexcitability of dorsal horn nociceptive neurons resulting in increased nociceptive transmission and pain perception. This state is associated with enhanced nociceptive signaling, spinal glutamate-mediated N-methyl-d-aspartate receptor activation, neuroimmune activation, nitroxidative stress, and supraspinal descending facilitation. The nitroxidative species considered for their role in nociception and central sensitization include nitric oxide (NO), superoxide ( \text O₂^·-{\text {O}_2}^{{\cdot }^{-}}), and peroxynitrite (ONOO⁻). Nitroxidative species are implicated during persistent but not normal nociceptive processing. This review examines the role of nitroxidative species in pain through a discussion of their contributions to central sensitization and the underlying mechanisms. Future directions for nitroxidative pain research are also addressed. As more selective pharmacologic agents are developed to target nitroxidative species, the exact role of nitroxidative species in pain states will be better characterized and should offer promising alternatives to available pain management options.     

Tyrosine nitration by superoxide and nitric oxide fluxes in biological systems: modeling the impact of superoxide dismutase and nitric oxide diffusion

Tyrosine nitration is a posttranslational modification observed in many pathologic states that can be associated with peroxynitrite (ONOO(-)) formation. However, in vitro, peroxynitrite-dependent tyrosine nitration is inhibited when its precursors, superoxide (O(2)*(-)) and nitric oxide ((*)NO), are formed at ratios (O(2)*(-)/(*)NO) different from one, severely questioning the use of 3-nitrotyrosine as a biomarker of peroxynitrite-mediated oxidations. We herein hypothesize that in biological systems the presence of superoxide dismutase (SOD) and the facile transmembrane diffusion of (*)NO preclude accumulation of O(2)*(-) and (*)NO radicals under flux ratios different from one, preventing the secondary reactions that result in the inhibition of 3-nitrotyrosine formation. Using an array of reactions and kinetic constants, computer-assisted simulations were performed in order to assess the flux of 3-nitrotyrosine formation (J(NO(2(-))Y)) during exposure to simultaneous fluxes of superoxide (J(O(2)*(-))) and nitric oxide (J((*)NO)), varying the radical flux ratios (J(O(2)*(-))/ J((*)NO)), in the presence of carbon dioxide. With a basic set of reactions, J(NO(2(-))Y) as a function of radical flux ratios rendered a bell-shape profile, in complete agreement with previous reports. However, when superoxide dismutation by SOD and (*)NO decay due to diffusion out of the compartment were incorporated in the model, a quite different profile of J(NO(2(-))Y) as a function of the radical flux ratio was obtained: despite the fact that nitration yields were much lower, the bell-shape profile was lost and the extent of tyrosine nitration was responsive to increases in either O(2)*(-) or (*)NO, in agreement with in vivo observations. Thus, the model presented herein serves to reconcile the in vitro and in vivo evidence on the role of peroxynitrite in promoting tyrosine nitration.     

Circadian variation of nitric oxide synthase activity in mouse tissue

Endogenous nitric oxide (NO) is an important mediator in the processes that control biological clocks and circadian rhythms. The present study was designed to elucidate if NO synthase (NOS) activity in the brain, kidney, testis, aorta, and lungs and plasma NO_x levels in mice are controlled by an endogenous circadian pacemaker. Male BALB/c mice were exposed to two different lighting regimens of either light-dark 14:10 (LD) or continuous lighting (LL). At nine different equidistant time points (commencing at 09:00h) blood samples and tissues were taken from mice. The plasma and tissue homogenates were used to measure the levels of NO₂+ NO₃^- (NO_x) and total protein. The NO_x concentrations were determined by a commercial nitric oxide synthase assay kit, and protein content was assessed in each homogenate tissue sample by the Lowry method. Nitric oxide synthase activity was calculated as pmol/mg protein/h. The resulting patterns were analyzed by the single cosinor method for pre-adjusted periods and by curve-fitting programs to elucidate compound rhythmicity. The NOS activity in kidneys of mice exposed to LD exhibited a circadian rhythm, but no rhythmicity was detected in mice exposed to LL. Aortic NOS activity displayed 24h rhythmicity only in LL. Brain, testis, and lung NOS activity and plasma NO_x levels displayed 24h rhythms both in LD and LL. Acrophase values of NOS activity in brain, kidney, testis, and lungs were at midnight corresponding to their behavioral activities. Compound rhythms were also detected in many of the examined patterns. The findings suggest that NOS activity in mouse brain, aorta, lung, and testis are regulated by an endogenous clock, while in kidney the rhythm in NOS activity is synchronized by the exogenous signals.     

Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.     

Protective roles of nitric oxide on germination and antioxidant metabolism in wheat seeds under copper stress

Nitric oxide (NO) is a multifunctional gaseous signal in plant. In the present study, we found that pretreatment with NO could significantly improve wheat seeds germination and alleviate oxidative stress against copper toxicity. With the enhancement of copper stress, the germination percentage of wheat seeds decreased gradually. Pretreatment during wheat seed imbibition with sodium nitroprusside (SNP), an NO donor, could greatly reverse the inhibitory effect of the following copper stress to wheat seeds germination. SNP-pretreated seeds also tended to retain higher amylase activities than that of the control without SNP pretreatment. On the other hand, there was no apparent difference in the activities of esterase in wheat seeds pretreated with or without SNP. Further investigations showed that pretreatment with NO donor dramatically stimulated the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), decreased the activities of lipoxygenases, sustained a lower level of malondialdehyde, and interfered with hydrogen peroxide (H₂O₂) excessive accumulation compared with the control, thereby enhancing the antioxidative capacity in wheat seeds under copper stress. In addition, the seed copper contents were not significant different between those pretreated with SNP and the controls, inferring that protective roles of NO was not responsible for preventing Cu uptake. Kang-Di Hu and Lan-Ying Hu contributed equally to this paper.     

Involvement of nitric oxide in noradrenaline-induced changes in the activity of antioxidant enzymes and lipid peroxidation in rat brown adipose tissue and heart

The effect of exogenous noradrenaline (NA) (1.6 mg.kg(-1) i.p., 35 min prior sacrifice) on the activity of antioxidant enzymes (AOE) copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT), as well as lipid peroxides (LP) concentration were studied in the rat interscapular brown adipose tissue (IBAT) and heart of saline (controls) and N(omega)-nitro-L-arginine methyl ester (L-NAME) treated rats (10 mg.kg(-1), i.p., during 3 days and 20 min before NA). NA differently affects both AOE activities and LP production in the IBAT and heart. Thus, NA inhibited the activity of all IBAT AOE and LP production while in the heart it markedly increased CAT activity only, but had no effect on any of SODs activities and LP concentration. L-NAME, a nitric oxide synthase blocker, completely abolished the NA-induced inhibition of the IBAT AOE and LP production, whereas in the heart it was without effect. In conclusion, these results indicate that both NA and L-NAME effects on AOE activity and LP production are tissue specific and also suggest that nitric oxide mediates the NA-induced inhibition of AOE activity and LP production in the IBAT only.     

Opposing interplay between Neuropeptide FF and nitric oxide in antinociception and hypothermia

This study examined the ability of the anti-opioid Neuropeptide FF (NPFF) to modify the endogenous activity of nitric oxide (NO). Antinociceptive and hypothermic effects of 1DMe (D.Tyr-Leu-(n.Me)Phe-Gln-Pro-Gln-Arg-Phe-NH(2)), an NPFF agonist, and of L-NAME (N(omega)nitro-L-arginine methyl ester), an inhibitor of nitric oxide synthase, were investigated in mice. Intraperitoneal (i.p.) injection of L-NAME induced, in the hot plate test, a dose-dependent antinociception not reversed by naloxone, an opioid antagonist, but inhibited by L-Arg, the NO synthesis precursor. Intracerebroventricular (i.c.v.) injections of 1DMe inhibit the antinociceptive activity of L-NAME in a dose-dependent manner. On the contrary, L-NAME markedly potentiated hypothermia induced by 1DMe injected in the third ventricle. These data show that Neuropeptide FF receptors exert a dual effect on endogenous NO functions and could modulate pain transmission independently of opioids.     

Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia

Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O(2)(-) balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO(2) was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O(2)(-) were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and N(omega)-nitro-L-arginine methyl ester (L-NAME) administration on NO and O(2)(-) were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O(2) (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO(2) was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O(2)(-)-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. L-NAME treatment reduced O(2)(-) relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O(2)(-) relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O(2)(-), and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O(2)(-). The finding that tetrahydrobiopterin, apocynin, and L-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.     

Opposing roles of EGF in IFN-alpha-induced epithelial barrier destabilization and tissue repair

Balance between damaging influences and repair mechanisms determines the degree of tissue deterioration by inflammatory and other injury processes. Destabilization of the proximal tubular barrier has been previously shown to be induced by IFN-alpha, a cytokine crucial for linking innate and adaptive immune responses. EGF was implicated in rescue mechanisms from renal injury. To study the interplay between the two processes, we determined if EGF can prevent IFN-alpha-induced barrier permeabilization. EGF did not counteract but even exacerbated the IFN-alpha-induced decrease of transepithelial electrical resistance in LLC-PK1 monolayers. For this effect Erk1/2 activation was necessary, linking barrier regulation to EGF-induced cell cycle progression. In contrast to its damage-intensifying effect, EGF also facilitated the regeneration of epithelial barrier function after the termination of IFN-alpha treatment. This effect was not mediated by Erk1/2 activation or cell proliferation since U0126, an Erk1/2 inhibitor, did not prevent but ameliorated recovery. However, EGF accelerated the downregulation of caspase-3 in recovering cells. Similarly, a pan-caspase inhibitor was able to block caspase activity and, concomitantly, promote restoration of barrier function. Thus, barrier repair might be linked to an EGF-mediated antiapoptotic mechanism. EGF appears to sensitize epithelial cells to the detrimental effects of IFN-alpha but also helps to restore barrier function in the healing phase. The observed dual effect of EGF might be explained by the different impact of proproliferative and antiapoptotic signaling pathways during and after cytokine treatment. The timing of epithelial exposure to damaging agents and repair factors was identified as a crucial parameter determining tissue fate.     

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.