Central nervous system, peripheral and hemodynamic effects of nanoformulated anandamide in hypertension.

PURPOSE: Hypertensive lesions induce alterations at hemodynamic, peripheral, and central levels. Anandamide (N-arachidonoylethanolamine; AEA) protects neurons from inflammatory damage, but its free administration may cause central adverse effects. AEA controlled release by nanoformulations could reduce/eliminate its side effects. The present study aimed to evaluate the effects of nanoformulated AEA (nf-AEA) on systolic blood pressure (SBP), behavior, and central/peripheral inflammatory, oxidative, and apoptotic state in spontaneously hypertensive rats (SHR). MATERIALS/METHODS: Male rats were used, both Wistar Kyoto (WKY) and SHR (n â€‹= â€‹10 per group), with/without treatment with nf-AEA (obtained by electrospraying) at a weekly dose of 5 â€‹mg/kg IP for 4 weeks. SBP was measured and behavioral tests were performed. Inflammatory/oxidative markers were quantified at the central (brain cortex) and peripheral (serum) level. RESULTS: SHR showed hyperactivity, low anxiety, and high concentrations of central/peripheral inflammatory/oxidative markers, also higher apoptosis of brain cortical cells compared to WKY. As opposed to this group, treatment with nf-AEA in SHR significantly reduced SBP, peripheral/central inflammatory/oxidative makers, and central apoptosis. Nf-AEA also increased neuroprotective mechanisms mediated by intracellular heat shock protein 70 (Hsp70), which were attenuated in untreated SHR. Additionally, nf-AEA reversed the abnormal behaviors observed in SHR without producing central adverse effects. CONCLUSIONS: Our results suggest protective properties of nf-AEA, both peripherally and centrally, through a signaling pathway that would involve the type I angiotensin II receptor, Wilms tumor transcription factor 1, Hsp70, and iNOS. Considering non-nf-AEA limitations, this nanoformulation could contribute to the development of new antihypertensive and behavioral disorder treatments associated with neuroinflammation.

Allopregnanolone reversion of estrogen and progesterone memory impairment: interplay with serotonin release.

Previously, we found out that in ovariectomized female rats, estrogen and progesterone produce a memory deficit which is reverted by the intrahippocampal administration of allopregnanolone. Here, we study the possible interplay between allopregnanolone and hippocampal serotonergic activity. Ovariectomized rats injected subcutaneously with estrogen and progesterone were subsequently injected in the dorsal hippocampus with vehicle, allopregnanolone alone or allopregnanolone shortly after 8OH-DPAT, a predominantly 5HT1A-7 receptor agonist. Then, the subjects were sequentially tested in: (1) an inhibitory avoidance task and (2) K+-evoked [3H]-serotonin ex vivo release through superfusion experiments. Allopregnanolone increased the K+-evoked [3H]-serotonin release compared to control. 8OH-DPAT infusions reversed the effects of allopregnanolone on memory and K+-evoked [3H]-serotonin release. These results suggest that allopregnanolone memory improvement could be mediated, at least in part, through modulation of the hippocampal serotonergic system reactivity.

Progesterone Exerts a Neuromodulatory Effect on Turning Behavior of Hemiparkinsonian Male Rats: Expression of 3 α -Hydroxysteroid Oxidoreductase and Allopregnanolone as Suggestive of GABAA Receptors Involvement.

There is a growing amount of evidence for a neuroprotective role of progesterone and its neuroactive metabolite, allopregnanolone, in animal models of neurodegenerative diseases. By using a model of hemiparkinsonism in male rats, injection of the neurotoxic 6-OHDA in left striatum, we studied progesterone's effects on rotational behavior induced by amphetamine or apomorphine. Also, in order to find potential explanatory mechanisms, we studied expression and activity of nigrostriatal 3α-hydroxysteroid oxidoreductase, the enzyme that catalyzes progesterone to its active metabolite allopregnanolone. Coherently, we tested allopregnanolone for a possible neuromodulatory effect on rotational behavior. Also, since allopregnanolone is known as a GABAA modulator, we finally examined the action of GABAA antagonist bicuculline. We found that progesterone, in addition to an apparent neuroprotective effect, also increased ipsilateral expression and activity of 3α-hydroxysteroid oxidoreductase. It was interesting to note that ipsilateral administration of allopregnanolone reversed a clear sign of motor neurodegeneration, that is, contralateral rotational behavior. A possible GABAA involvement modulated by allopregnanolone was shown by the blocking effect of bicuculline. Our results suggest that early administration of progesterone possibly activates genomic mechanisms that promote neuroprotection subchronically. This, in turn, could be partially mediated by fast, nongenomic, actions of allopregnanolone acting as an acute modulator of GABAergic transmission.

Postnatal administration of allopregnanolone modifies glutamate release but not BDNF content in striatum samples of rats prenatally exposed to ethanol.

Ethanol consumption during pregnancy may induce profound changes in fetal CNS development. We postulate that some of the effects of ethanol on striatal glutamatergic transmission and neurotrophin expression could be modulated by allopregnanolone, a neurosteroid modulator of GABAA receptor activity. We describe the acute pharmacological effect of allopregnanolone (65 µg/kg, s.c.) administered to juvenile male rats (day 21 of age) on the corticostriatal glutamatergic pathway, in both control and prenatally ethanol-exposed rats (two ip injections of 2.9 g/kg in 24% v/v saline solution on gestational day 8). Prenatal ethanol administration decreased the K(+)-induced release of glutamate regarding the control group. Interestingly, this effect was reverted by allopregnanolone. Regarding BDNF, allopregnanolone decreases the content of this neurotrophic factor in the striatum of control groups. However, both ethanol alone and ethanol plus allopregnanolone treated animals did not show any change regarding control values. We suggest that prenatal ethanol exposure may produce an alteration of GABAA receptors which blocks the GABA agonist-like effect of allopregnanolone on rapid glutamate release, thus disturbing normal neural transmission. Furthermore, the reciprocal interactions found between GABAergic neurosteroids and BDNF could underlie mechanisms operating during the neuronal plasticity of fetal development.

Neuromodulatory effect of progesterone on the dopaminergic, glutamatergic, and GABAergic activities in a male rat model of Parkinson's disease.

OBJECTIVES: Progesterone has been reported to have a neuroprotective role in depression-like rats in a hemiparkinsonian model of the disease. In this work, we investigate if this hormone affects the three principal neurochemicals striatal systems (dopaminergic, glutamatergic, and GABAergic) that are involved in the physiopathology of the disease in a hemiparkinsonim male rat model at 8 weeks post-chemical injury. METHODS: For this purpose, we design three experimental groups: (1) sham group; (2) hemiparkinsonian group; and (3) hemiparkinsonian group subcutaneously injected with progesterone at 7 days post-chemical injury. Animals were tested in an automated rotational device at 8 weeks post-chemical injury. After behavioral test, K(+)-evoked [(3)H]-dopamine, [(3)H]-glutamate, and [(3)H]-gamma aminobutyric acid release from striatum slices were analyzed by superfusion experiments. RESULTS: The hemiparkinsonian group showed distinctive alterations that are produced by neurodegeneration of left nigrostriatal dopaminergic pathway by 6-hydroxydopamine hydrobromide (6-OHDA). On the other hand, the administration of progesterone 7 days after the injection of the neurotoxin was able to (1) improve the K(+)-evoked [(3)H]-dopamine release from the damaged striata (left); (2) avoid significant increase in the K(+)-evoked [(3)H]-glutamate release from the left striata; and (3) progesterone does not modify the K(+)-evoked [(3)H]-gamma aminobutyric acid release from the left striata. DISCUSSION: These results suggest that progesterone does have neuroprotective and neuromodulatory effects on striatal neurotransmission systems in the hemiparkinsonian male rats. The possible mechanisms would involve genomic and non-genomic actions of this neuroactive steroid which would modulate the activity of dopaminergic, glutamatergic, and GABAergic pathways.

Allopregnanolone prevents memory impairment: effect on mRNA expression and enzymatic activity of hippocampal 3-α hydroxysteroid oxide-reductase.

In this work we investigated how the neurosteroid allopregnanolone can modulate learning and memory processes. For this purpose, we used ovariectomized (OVX) rats subcutaneously injected with oestradiol benzoate (E) alone or E and progesterone (P). Then, rats were injected in dorsal hippocampus with allopregnanolone or vehicle. Animals were tested in inhibitory avoidance task (IA task). After behavioural test hippocampal mRNA expression and enzymatic activity of 3α-HOR, the enzyme responsible of allopregnanolone synthesis, were analysed. In IA task OVX-EP rats spent less time on platform, compared to those OVX or OVX-E. Regression analyses revealed that there was a significant negative relationship between E-P infusion and performance in this task. Pre-training allopregnanolone administration to OVX-EP rats increased the time spent on the platform. Interestingly, when enzymatic activity of 3α-HOR was tested, OVX-EP rats showed a significant decrease in the enzymatic activity, compared with OVX and OVX-E rats. In addition, OVX-EP group showed a significant increase in the enzymatic activity after intrahippocampal infusion of allopregnanolone. On the other hand, when mRNA expression of 3α-HOR was analysed no differences were observed when the hippocampal allopregnanolone injection was done. These results suggest that E and P have amnesic effects on female rats, being reversed by allopregnanolone through its modulation on hippocampal 3α-HOR activity.

Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis.

Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc.

Progesterone prevents depression-like behavior in a model of Parkinson's disease induced by 6-hydroxydopamine in male rats.

Hemiparkinsonism induced by 6-hydroxydopamine (6-OHDA) injected in left corpus striatum is a recognized model of motor deficits in rats. Some reports concerning motor deficits indicate a favorable response to steroid administration in hemiparkinsonian animals. However, there is no much information regarding progesterone administration in relation to cognitive and affective dysfunctions. Here we could confirm earlier reports regarding a mild deficit of memory and a noticeable depressive-like behavior 4 weeks after injecting 6-OHDA. We also present some evidence that progesterone could be - when administered 7 days after the injection of 6-OHDA - a possible neuroprotector concerning both motor deficits as well as cognitive - memory- and depression-like behaviors. The affective deficit was reverted by administering the tricyclic antidepressant imipramine. Since Parkinson's disease is a conspicuous cause of psycho-organic decline in human beings, it would be important to be able of dealing early with non-motor indicators in order to use prospective neuroprotectors to prevent the progression of the disease.

Allopregnanolone induces LHRH and glutamate release through NMDA receptor modulation.

LHRH release from hypothalamus is influenced by the neurotransmitter glutamate that acts, among others, on NMDA receptors present in LHRH neurons. On the other hand, the neurosteroid allopregnanolone can modulate the activity of specific neurotransmitter receptors and affect neurotransmitter release. We examined the role of allopregnanolone on in vitro LHRH and glutamate release from mediobasal hypothalamus and anterior preoptic area of ovariectomized rats with estrogen and progesterone replacement. Moreover, we evaluated whether the neurosteroid might act through modulation of NMDA receptors. Allopregnanolone induced an increase in LHRH release. This effect was reversed when the NMDA receptors were blocked by the NMDA antagonist 2-amino-7-phosphonoheptanoic acid (AP-7) indicating that this neurosteroid would interact with NMDA receptors. Moreover allopregnanolone induced an augment in K(+) evoked [(3)H]-glutamate release from mediobasal hypothalamus-anterior preoptic area explants and this effect was also reversed when NMDA receptors were blocked with AP-7. These results suggest an important physiologic function of allopregnanolone on the regulation of neuroendocrine function in female adult rats. Not only appears to be involved in enhancing LHRH release through modulation of NMDA receptors but also in the release of glutamate which is critical in the control of LHRH release.

Pregnenolone sulfate infused in lateral septum of male rats impairs novel object recognition memory.

In the present paper we show for the first time that pregnenolone sulfate (Preg-S) impairs rats' memory for novel object recognition when injected in lateral septum (1.2 microM). The effect of Preg-S is clearly related to the moment the reagent is administered: if administered shortly after the training phase, or prior to the test phase of the experiment, there is no amnesic effect. It is only amnesic when administered 30 min before training. Accordingly, Preg-S does not appear to affect the storage of new memories or their retrieval but rather the acquisition itself. Based on the described afferences and efferences of lateral septum, we suggest a possible stimulatory effect of Preg-S regarding glutamate receptors and/or an inhibitory effect of GABA receptors located in local interneurons or recurrent axon collaterals, both of which have been reported to exist in the aforementioned nucleus.

Cyclic nucleotide phosphodiesterase inhibition increases tyrosine phosphorylation and hyper motility in normal and pathological human spermatozoa

Our objective was to determine the effect of phosphodiesterase (PDE) inhibition on: 1) tyrosine phosphorylation of human spermatozoa at the tail level; and 2) sperm motion parameters and hyperactivated motility. The study was conducted with normozoospermic and asthenozoospermic samples incubated under in vitro capacitating conditions. The main outcome measures were computer-assisted sperm motion analysis and fluorescent immunodetection of phosphotyrosine-containing proteins. Pentoxifylline (PTX) was used as PDE inhibitor because of its wide use in the clinic. PTX-treatment significantly increased sperm velocity, hyperactivated motility and tyrosine-phosphorylation, both in normo and asthenozoospermic samples. Tyrosine-phosphorylation of tail proteins was highly conspicuous in both types of samples, showing no differential pattern after PTX-treatment. Normozoospermic samples treated with pentoxifylline showed an increase in the number of spermatozoa displaying hyperactivated movement and tyrosine-phosphorylation at the tail level. Preliminary data on asthenozoospermic samples exhibiting altered motion characteristics and defective phosphorylation of sperm-tail proteins showed that both defects can be concomitantly overcome by pentoxifylline treatment. Tyrosine-phosphorylation of sperm-tail proteins is underlying the enhancement of hyperactivated motility resulting from PDE inhibition by pentoxifylline.

Cyclic nucleotide phosphodiesterase inhibition increases tyrosine phosphorylation and hyper motility in normal and pathological human spermatozoa.

Our objective was to determine the effect of phosphodiesterase (PDE) inhibition on: 1) tyrosine phosphorylation of human spermatozoa at the tail level; and 2) sperm motion parameters and hyperactivated motility. The study was conducted with normozoospermic and asthenozoospermic samples incubated under in vitro capacitating conditions. The main outcome measures were computer-assisted sperm motion analysis and fluorescent immunodetection of phosphotyrosine-containing proteins. Pentoxifylline (PTX) was used as PDE inhibitor because of its wide use in the clinic. PTX-treatment significantly increased sperm velocity, hyperactivated motility and tyrosine-phosphorylation, both in normo and asthenozoospermic samples. Tyrosine-phosphorylation of tail proteins was highly conspicuous in both types of samples, showing no differential pattern after PTX-treatment. Normozoospermic samples treated with pentoxifylline showed an increase in the number of spermatozoa displaying hyperactivated movement and tyrosine-phosphorylation at the tail level. Preliminary data on asthenozoospermic samples exhibiting altered motion characteristics and defective phosphorylation of sperm-tail proteins showed that both defects can be concomitantly overcome by pentoxifylline treatment. Tyrosine-phosphorylation of sperm-tail proteins is underlying the enhancement of hyperactivated motility resulting from PDE inhibition by pentoxifylline.

Incidence of sperm-tail tyrosine phosphorylation and hyperactivated motility in normozoospermic and asthenozoospermic human sperm samples.

Our objective was to study the incidence of sperm-tail phosphotyrosine immunoreactivity in normozoospermic and asthenozoospermic human sperm samples, its association with sperm motion parameters, particularly hyperactivated motility, and its potential involvement in the pathogenesis of asthenozoospermia. The work was conducted as a prospective experimental study in the Sperm Biology and Andrology laboratories of the Jones Institute, a medical school-based fertility center. The study subjects were healthy fertile male donors (normozoospermic samples) and infertile patients (asthenozoospermic samples) attending the center. Recently ejaculated semen samples were washed twice to eliminate seminal plasma and a swim-up was performed to select the motile population which, in turn, was incubated up to 18 h at 37 degrees C in 3.5% human serum albumin-supplemented Ham's F10 to allow for capacitation. For evaluation, sperm aliquots were taken pre-swim-up (T0), immediately post swim-up (T1), at 6 h (T6), and 18 h (T18) of incubation. The main outcome measures were computer-analyzed sperm motion parameters and hyperactivated motility, and immunodetection of phosphotyrosine (PY)-containing proteins. During the capacitating incubation, normozoospermic samples displayed maximum motility, velocity, and hyperactivation at T6, significantly decreasing their values at T18. PY-proteins were located both at the tail and head of spermatozoa. Their expression increased progressively during the incubation, being present in about 70% of the sperm tails at T18. Asthenozoospermic samples showed an inability to respond to capacitation with an increase in motion parameters and PY-phosphorylation. At T6, both hyperactivation and PY-phosphorylation were significantly lower than in normal samples. Our results suggest that PY-phosphorylation of tail proteins is highly conspicuous in human spermatozoa, and increases its incidence in a time-dependent manner, as more sperm become capacitated. Asthenozoospermic samples displaying low percentages of motile sperm and altered motion characteristics showed a decreased incidence of PY-phosphorelated sperm. Tail protein PY-phosphorylation may be related to sperm movement, especially to hyperactivated motility and its deficiency may be associated to asthenozoospermia.

The intraacrosomal calcium pool plays a direct role in acrosomal exocytosis.

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP(3)-sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP(3)-sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.