José A. Rodrigo

Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats

Two NO synthase (NOS) isoforms have been described in vessels, an endothelial constitutive NOS (eNOS) and an inducible NOS (iNOS). The purpose of the present study was to examine the endothelium-dependent and endothelium-independent hypotensive response in aging rats, analyzing the ability of their vessels to produce NO. The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (n=20, 18 months old) and young rats (n=20, 5 months old). The hypotensive responses to acetylcholine, bradykinin, and sodium nitroprusside were determined. Furthermore, the expression of the NOS isoforms by Western blot and the eNOS and iNOS activities, defined as Ca2+-dependent and Ca2+-independent conversion of [14C]L-arginine into [14C]L-citrulline, respectively, were also determined. In the aging rats, we found an impaired hypotensive response to acetylcholine and bradykinin (2 NO- and endothelium-dependent hypotensive agents) that was accompanied by a preserved hypotensive response to sodium nitroprusside. Aging rats also demonstrated an enhanced sensitivity response to the pressor effect of the L-arginine antagonist L-Nomega-nitro-L-arginine and a reduced vasoconstrictor response to angiotensin II. The inhibition of NO synthesis normalized the pressor effect of angiotensin II in the aging animals. Nitrite plus nitrate plasma levels were increased in aging rats. Furthermore, cGMP content was also higher in the aging vessels. In the aging aortas, the expression of both eNOS and iNOS isoforms was enhanced. However, in aging rats, the activity of the eNOS isoform was markedly reduced, a finding that was accompanied by the presence of iNOS activity. The vessel wall of aging rats showed an enhanced expression of eNOS and iNOS isoforms. However, eNOS activity was reduced in the aging animals. These findings could explain the impaired endothelium-dependent hypotensive response associated with aging.

Glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain.

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurodegenerative disorders. We have previously demonstrated that chronic stress induced an increase in nitric oxide (NO) production via an expression of inducible NO synthase (iNOS) in brain. Since it has been demonstrated that NO regulates mitochondrial function, we sought to study the susceptibility of the mitochondrial respiratory chain complexes to chronic restrain stress exposure in brain cortex. In adult male rats, stress (immobilization for six hours during 21 days) inhibits the activities of the first complexes of the mitochondrial respiratory chain (inhibition of 69% in complex I-III and of 67% in complex II-III), without affecting complex IV activity, ATP production and oxygen consumption. The mitochondrial marker citrate synthase is not significantly affected by stress after 21 days, indicating that at this time the mitochondrial structure is still intact. Moreover, the administration of the preferred inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) protects against the inhibition of the activity of complexes of the mitochondrial respiratory chain as well as prevents NOx− accumulation, lipid peroxidation and glutathione depletion induced by stress. These results suggest that a sustained overproduction of NO via iNOS is responsible, at least in part, of the inhibition of mitochondrial respiratory chain caused by stress and that this pathway also accounts for the oxidative stress found in this situation.

Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus

NO is a widespread messenger molecule in physiology. We were interested in investigating whether an NO‐generating system could be present in plants. NO and l‐[14C]citrulline were synthesized by roots and nodules of Lupinus albus in an l‐arginine‐dependent manner. l‐[14C]Citrulline production was inhibited by NG‐monomethyl‐l‐arginine, a nitric oxide synthase antagonist, in a competitive way. NADPH‐diaphorase activity was localized in the vascular bundles in root and nodules, and also in the nodule infected zone. This staining was significantly reduced in the presence of NG‐monomethyl‐l‐arginine. These results indicate the presence of a putative nitric oxide synthase in plants.

Chronic Stress Induces the Expression of Inducible Nitric Oxide Synthase in Rat Brain Cortex

Abstract: Long‐term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium‐independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2‐ + NO3‐ (NOx‐) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx‐ accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.

Selective nitrergic neurodegeneration in diabetes mellitus–a nitric oxide‐dependent phenomenon

In vitro and in vivo studies have demonstrated a dysfunctional nitrergic system in diabetes mellitus, thus explaining the origin of diabetic impotence. However, the mechanism of this nitrergic defect is not understood. In the penises of streptozotocin (STZ)‐induced diabetic rats, here, we show by immunohistochemistry that nitrergic nerves undergo selective degeneration since the noradrenergic nerves which have an anti‐erectile function in the penis remained intact. Nitrergic relaxation responses in vitro and erectile responses to cavernous nerve stimulation in vivo were attenuated in these animals, whereas noradrenergic responses were enhanced. Activity and protein amount of neuronal nitric oxide synthase (nNOS) were also reduced in the penile tissue of diabetic rats. We, thus, hypothesized that NO in the nitrergic nerves may be involved in the nitrergic nerve damage, since only the nerves which contain neuronal NO synthase underwent degeneration. We administered an inhibitor of NO synthase, NG‐nitro‐L‐arginine methyl ester (L‐NAME), in the drinking water of rats for up to 12 weeks following the establishment of diabetes with STZ. Here we demonstrate that this compound protected the nitrergic nerves from morphological and functional impairment. Our results show that selective nitrergic degeneration in diabetes is NO‐dependent and suggest that inhibition of NO synthase is neuroprotective in this condition.

Gyrator transform: properties and applications

In this work we formulate the main properties of the gyrator operation which produces a rotation in the twisting (position - spatial frequency) phase planes. This transform can be easily performed in paraxial optics that underlines its possible application for image processing, holography, beam characterization, mode conversion and quantum information. As an example, it is demonstrated the application of gyrator transform for the generation of a variety of stable modes.

Neuronal and inducible nitric oxide synthase and nitrotyrosine immunoreactivities in the cerebral cortex of the aging rat.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS‐immunoreactive multipolar neurons found in layers II–VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS‐positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II–VI of aged rat cortex. While no iNOS‐immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine‐positive cells outnumbered total NOS‐positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine‐positive cells. Western blots of brain extracts showed a several‐fold increase in both nNOS‐ and iNOS‐immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine‐containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation. Microsc. Res. Tech. 43:75–88, 1998.

Induction of Cyclooxygenase-2 Accounts for Restraint Stress-Induced Oxidative Status in Rat Brain

Cyclooxygenase (COX) is the rate-limiting enzyme in the metabolism of arachidonic acid into prostanoids. Although it is constitutively expressed in brain neurons, the inducible isoform (COX-2) is also upregulated in pathological conditions such as seizures, ischemia or some degenerative diseases. To assess whether COX-2 is regulated after stress, we have used adult male Wistar rats, some of which were immobilized during 6 h. An increase in PGE2 concentration occurs in brain cortex after 2–6 h of the onset of stress as well as an enhancement of COX-2 protein. Immunohistochemical studies indicate that COX-2 is expressed in the cortex and hippocampus after stress in cells with morphology of neurons. Administration of PDTC (150 mg/kg), an inhibitor of the transcription factor NF-κB or MK-801 (0.2 mg/kg), an N-methyl-D-aspartate receptor blocker, prevents both stress-induced increase in COX-2 activity and protein levels, suggesting an implication of these factors in the mechanism by which stress induces COX-2 in brain. To assess if COX-2 accounts for the oxidative status seen in brain after stress, a group of animals were i.p. injected with NS-398, a specific COX-2 inhibitor 1 h prior to the onset of stress. NS-398 (5 mg/kg) decreases stress-induced malondialdehyde accumulation in cortex as well as prevents the stress-induced oxidation of glutathione. Finally, NS-398 reduced Ca2+-independent inducible nitric oxide synthase (iNOS, NOS-2) activity and lowered the stress-induced accumulation of NO metabolite levels in cortex. These effects of NS-398 seem to be due to the specific inhibition of COX-2, since it has no effect on stress-induced corticosterone release, glutamate release, and NF-κB activation. These findings are discussed as possible damaging and/or adaptive roles for stress-induced COX-2 in the brain.

Neuronal expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices

Nitric oxide (NO) overproduction has been postulated to contribute significantly to ischaemia‐reperfusion neurotoxicity. Inducible or type II NO synthase (iNOS) synthesizes NO in large quantities for long periods of time. Therefore we investigated the expression and localization of iNOS after oxygen and glucose deprivation in rat forebrain slices. In this experimental model, calcium‐independent NOS activity reached a maximum 180 min after the end of a 20 min oxygen–glucose deprivation period. During the same period of time, the calcium‐independent activity was absent in control forebrain slices. To test whether this calcium‐independent NOS activity was due to the expression of iNOS, the effects of the addition of dexamethasone, cycloheximide and pyrrolidine dithiocarbamate were determined. All of them inhibited the induction of the calcium‐independent NOS activity measured in the rat forebrain slices after oxygen and glucose deprivation. Furthermore, oxygen and glucose deprivation caused the expression of the gene encoding iNOS in rat forebrain slices, as assessed by the detection of iNOS message and protein in these samples. A sixfold increase in the iNOS mRNA levels was observed at 180 min and the time‐course of the expression of iNOS mRNA was in agreement with the temporal profile of iNOS enzymatic activity. Immunohistochemistry analysis revealed that iNOS was highly expressed in neurones, astrocytes and microglial cells. These results demonstrate for the first time that iNOS is expressed in neurones after oxygen and glucose deprivation, and that this expression occurs in short periods of time. These findings suggest that NO can play an important pathogenic role in the tissue damage that occurs after cerebral ischaemia.

Experimental implementation of the gyrator transform

The gyrator transform (GT) promises to be a useful tool in image processing, holography, beam characterization, mode transformation, and quantum information. We introduce what we believe to be the first flexible optical experimental setup that performs the GT for a wide range of transformation parameters. The feasibility of the proposed scheme is demonstrated on the gyrator transformation of Hermite-Gaussian modes. For certain parameters the output mode corresponds to the Laguerre-Gaussian one.