Antagonistic interactions between dexamethasone and fluoxetine modulate morphodynamics and expression of cytokines in astrocytes.

The "plasticity hypothesis" proposes that major depression is caused by morphological and biochemical modifications in neurons and astrocytes and those beneficial pharmacological effects of selective-serotonin-reuptake-inhibitors (SSRI) are at least partially associated with modifications of cellular communications between these cells. In this study we examined effects of the antidepressant fluoxetine on cultured astrocytes that were, in some cases, pretreated with dexamethasone, a cortisol analog known to trigger depressive disorder. Primary rat astrocytes were purified and treated with dexamethasone and the SSRI fluoxetine in physiological concentrations so that both drugs did not affect cell viability. Expression of interleukin-2 (IL-2) and glia-derived-neurotrophic-factor (GDNF) were analyzed and monitored and cell viability, apoptosis, cluster formation, particle-removing capacity and cell mobility were also monitored. Pre-studies without any drugs on mixed neuron-astrocyte co-cultures suggested that astrocytes interacted with neurons and other brain cells in vitro by actively assembling them into clusters. Treatment of purified astrocytes with dexamethasone significantly decreased their mobility compared to controls but had no effect on cluster formation. Dexamethasone-treated cells removed fewer extracellular particles derived from dead cells and cell debris. Both effects were abolished by simultaneous application of fluoxetine. Intracellular IL-2 increased, while GDNF amount expression was diminished following dexamethasone treatment. Simultaneous administration of fluoxetine reversed dexamethasone-triggered IL-2 elevation but had no effect on decreased GDNF concentration. These results suggest that mobility and growth factor equilibrium of astrocytes are affected by dexamethasone and by fluoxetine and that fluoxetine could reverse some changes induced by dexamethasone.

Expression of estrogen receptor α and ß in rat astrocytes in primary culture: effects of hypoxia and glucose deprivation.

Estrogen replacement therapy could play a role in the reduction of injury associated with cerebral ischemia in vivo, which could be, at least partially, a consequence of estrogen influence of glutamate buffering by astrocytes during hypoxia/ischemia. Estrogen exerts biological effects through interaction with its two receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), which are both expressed in astrocytes. This study explored effects of hypoxia and glucose deprivation (HGD), alone or followed by 1 h recovery, on ERα and ERß expression in primary rat astrocyte cultures following 1 h exposure to: a) 5 % CO(2) in air (control group-CG); b) 2 % O(2)/5 % CO(2) in N(2) with glucose deprivation (HGD group-HGDG); or c) the HGDG protocol followed by 1 h CG protocol (recovery group-RG). ERα mRNA expression decreased in HGDG. At the protein level, full-length ERα (67 kDa) and three ERα-immunoreactive protein bands (63, 60 and 52 kDa) were detected. A significant decrease in the 52 kDa band was seen in HGDG, while a significant decrease in expression of the full length ERα was seen in the RG. ERß mRNA and protein expression (a 54 kDa single band) did not change. The observed decrease in ERα protein may limit estrogen-mediated signalling in astrocytes during hypoxia and recovery.

Age-related changes in choroid plexus and blood-cerebrospinal fluid barrier function in the sheep.

Dysfunction of the choroid plexuses (CPs) and the blood-cerebrospinal fluid barrier (BCSFB) might contribute to age-related cognitive decline and neurodegenerative disease. We used the CPs from young (1-2 years), middle-aged (3-6 years) and old (7-10 years) sheep to explore effects of aging on various aspects of CP and BCSFB functions. Total protein in the cerebrospinal fluid (CSF) was significantly higher in old compared to young sheep and CSF secretion by the CP perfused in situ was significantly lower in both old and middle-aged when compared to young sheep, which correlated with reduced (22)Na(+) uptake and efflux by the CP. Steady-state extractions of a low and medium size molecular weight extracellular space marker, (14)C-mannitol and (3)H-polyethylene glycol, respectively, were significantly higher in CPs from old compared to young animals; however, there was no significant difference in steady-state extraction of a high molecular weight marker, (125)I-bovine serum albumin. This indicates increased passive BCSFB permeability for small and medium sized molecules in old sheep. CP redox activity was significantly lower in the old animals as assessed by the MTT assay, however, there was no significant difference in ATP content and energy charge of the CP with age suggesting adequate baseline energy reserve capacity. These data indicate that normal aging processes alter protein content in the CSF, CSF secretion, integrity of the BCSFB and Na(+) flux in the epithelial layer, which could impact on CSF homeostasis and turnover.

Ontogenic profile of estrogen receptor alpha and beta mRNA and protein expression in fetal rat brain.

It has been shown that increased estrogen can down-regulate its receptor, but there is no data to determine if that mechanism acts in the fetal brain as a consequence of high maternal estrogen levels. The aim of this study was to explore the expression of estrogen receptor (ER) in the developing fetal brain at 16, 19 and 21 days gestation (dg). The results revealed that both ERalpha and ERbeta isoforms, and some of their variants, were present in rat fetal brain at the transcript level and at the protein level. PCR results showed that the amount of ERalpha and ERbeta mRNA did not change significantly between 16, 19 and 21 dg; however, changes in protein expression were apparent. Two bands were detected for ERalpha protein by immunoblotting: the expression of the 73 kDa band, relative to the expression of actin, decreased significantly between 16 and 21 dg, while expression of the 67 kDa band did not change. Multiple variants of ERbeta were detected, including wild type ERbeta, ERbeta2 and ERbetaDelta5; the amounts of all decreased significantly, relative to the amount of actin, between 16 and 21 dg. A decrease in protein expression of some of the ER variants without an equivalent decrease in the amount of mRNA suggests that high levels of estrogen might triggered posttranslational modifications of ER, including the ER ubiquitin-dependent proteolysis. Overall, these findings suggest that the fetal brain might be less responsive to estrogens during late pregnancy in the rat, thereby minimizing the known harmful effects of high levels of circulating maternal estrogens.

The kinetics of hypoxanthine efflux from the rat brain.

The brain efflux of radiolabelled hypoxanthine in the rat was rapid in the first minute after injection [K(eff)(i)=0.21+/-0.06 min(-1)], which was saturable with a V(max)=13.08+/-0.81 nM min(-1) g(-1), and a high K(m,app) (67.2+/-13.4 microM); the K(i,app) for inosine was 31.5+/-7.6 microM. Capillary depletion analysis indicated that hypoxanthine accumulates in neurons and glia with the time. From cross-inhibition studies with different purines and pyrimidines, it suggests that these molecules could also be important substrates for this carrier.

The characteristics of nucleobase transport and metabolism by the perfused sheep choroid plexus.

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (U(max)) for hypoxanthine and adenine, was 35.51+/-1.50% and 30.71+/-0.49% and for guanine, thymine and uracil was 12.00+/-0.53%, 13.07+/-0.48% and 12.30+/-0.55%, respectively with a negligible backflux, except for that of thymine (35.11+/-5.37% of the U(max)). HPLC analysis revealed that the purine nucleobase hypoxanthine and the pyrimidine nucleobase thymine can pass intact through the choroid plexus and enter the cerebrospinal fluid CSF so the lack of backflux for hypoxanthine was not a result of metabolic trapping in the cell. Competition studies revealed that hypoxanthine, adenine and thymine shared the same transport system, while guanine and uracil were transported by a separate mechanism and that nucleosides can partially share the same transporter. HPLC analysis of sheep CSF collected in vivo revealed only two nucleobases were present adenine and hypoxanthine; with an R(CSF/Plasma) 0.19+/-0.02 and 3.43+/-0.20, respectively. Xanthine and urate, the final products of purine catabolism, could not be detected in the CSF even in trace amounts. These results suggest that the activity of xanthine oxidase in the brain of the sheep is very low so the metabolic degradation of purines is carried out only as far as hypoxanthine which then accumulates in the CSF. In conclusion, the presence of saturable transport systems for nucleobases at the basolateral membrane of the choroidal epithelium was demonstrated, which could be important for the distribution of the salvageable nucleobases, adenine and hypoxanthine in the central nervous system.

The kinetics of tiazofurin uptake by the isolated perfused choroid plexus of the sheep.

Tiazofurin (TZF-beta-D-ribofuronosyl thiazole-4-carboxamide, NSC-286193) is a synthetic nucleoside analog with potent antitumor activity. Isolated choroid plexuses (CP) of sheep were perfused in situ and the uptake of [3H]-tiazofurin was determined in relation to the recovery of [14C]-mannitol by means of the paired indicator dilution technique. The maximal uptake of tiazofurin was 8.29 +/- 0.84% and was shown to be both carrier-mediated, sodium-dependent and inhibited by adenosine which suggests that it uses the carrier for endogenous nucleosides. However, the total tiazofurin uptake into the choroid plexus was negligible (0.93 +/- 1.97%) as a result of a high backflux, indicating that tiazofurin is not trapped within the cells of the CP to any significant degree. The kinetics for the uptake into the CP were more favorable than for its passage across the blood-brain barrier with a Km of 7.71 +/- 1.42 microM, a Vmax of 1.30 +/- 0.05 microM/min/g and a negligible constant of a free diffusion (Kd) which suggests that the CP/CSF route may act as an alternative pathway into the brain.

Endogenous nucleosides in the guinea-pig eye: analysis of transport and metabolites.

This study investigates the transport of endogenous nucleosides and deoxynucleosides from the capillaries of the eye into the aqueous humour and the lens using the in situ vascular eye perfusion technique in the guinea-pig. The transport of [3H] adenosine and [3H] thymidine across the blood-aqueous barrier proved to be very rapid with a volume of distribution after 4 minutes perfusion reaching 11.9+/-3.0% and 9.93+/-1.1%, respectively. However, the transport of [3H] guanosine and [3H] cytidine was slower, with volumes of distribution reaching only 3.38+/-0.58% and 4.8+/-1.41%. The values for the entry of deoxyadenosine and deoxyguanosine were not significantly different from the values obtained for corresponding ribonucleosides (adenosine and guanosine) so that a change in the pentose sugar does not change the affinity of the nucleoside for the transport protein. Perfusion with a low sodium medium inhibited the transport of [3H] adenosine and [3H] thymidine into the aqueous humour. The presence of 800 nM NBTI also caused a decrease in adenosine transport into the aqueous humour, so that the volume of distribution after 2 minutes reached only 3.78+/-1.87%. These findings suggest that the transfer of adenosine across the blood-aqueous barrier has both concentrative and equilibrative components. The presence of 0.1 mM thymidine had no effect on the [3H] adenosine transport, whereas 0.1 mM of adenosine resulted in a marked decrease on the [3H] thymidine transport which suggests that the concentrative nucleotide transport is probably mediated by both cif and cit transport systems. The cellular uptake of nucleosides into the lens was very rapid and the volume of distribution of purine nucleosides was within the range of 30-50% whereas that for thymidine uptake was somewhat lower, reaching 20-30%. HPLC analysis of the eye structures in the guinea-pig showed that lens, vitreous body and the rest of the eye do not contain either free nucleosides or purine bases in detectable quantities, except for xanthine which was detected in aqueous humour at a concentration of 2.51+/-0.51 mM. However, serum of the anaesthetised guinea-pig did not contain xanthine in detectable amount so it seems that the metabolic degradation of the nucleosides in the guinea-pig eye progresses as far as xanthine, which is then accumulated in the aqueous humour.

The characteristics of basolateral nucleoside transport in the perfused sheep choroid plexus and the effect of nitric oxide inhibition on these processes.

The single pass paired dilution technique was used to measure the uptake of nucleosides across the basolateral face of the isolated in situ perfused sheep choroid plexus (CP). The uptake of labelled adenosine and guanosine into the CP was large (approximately 35%) whereas that of thymidine was less (approximately 15%). The addition of 0.5 mM unlabelled adenosine to the perfusate inhibited the uptake of labelled adenosine by 66%, guanosine by 100% and that of thymidine by 50%, whereas the addition of 0.5 mM unlabelled thymidine caused complete self-inhibition. The backflux of adenosine was very small which may indicate a high rate of cellular metabolism or a flux into cerebrospinal fluid (CSF). The addition of 0.5 mM unlabelled adenosine did not alter the backflux of adenosine, but increased that of guanosine and thymidine. The entry of radioactivity derived from adenosine across the apical side of the CP cells into the newly formed CSF was determined as a 'CSF uptake index' relative to [14C]butanol and found to be about 25%; however, HPLC analysis revealed that the majority of this activity was hypoxanthine, and not adenosine. The complete inhibition of nitric oxide synthase caused a significant reduction in adenosine uptake into the CP and an increase in backflux for this molecule. It would appear that the uptake for adenosine by the CP is governed by the rate of cellular metabolism and not by the rate of transport into the cells of the choroid plexus whereas for guanosine and thymidine, transport is of greater importance.

Kinetics and sodium independence of [3H]-tiazofurin blood to brain transport in the guinea pig.

Tiazofurin is a nucleoside analog with potent antitumor activity. The objective of this study was to define the kinetic parameters of tiazofurin transport from blood into the guinea pig brain. The second aim was to define kinetic parameters of tiazofurin transport inhibition by adenosine (Ki and Kda), as well as whether tiazofurin blood-to-brain transport was performed in countertransport with Na+. In order to determine these parameters, the in situ method of perfusion of guinea pig brain was used. Addition of increasing concentrations of unlabelled tiazofurin to perfusing medium (0.05-2.7 mmol/l) caused progressive decrease of [3H]-tiazofurin brain clearance with Km values ranging from 119.57 +/- 40.12 to 150.17 +/- 51.68 x 10(-6) mol/l. Maximal transport capacity ranged from 325.03 +/- 113.93 to 417.50 +/- 151.53 pmol/min/g. Introduction of adenosine into the perfusing medium (0.005-0.2 mmol/l) caused similar but more rapid decrease of [3H]-tiazofurin brain clearance (Ki = 6.36 +/- 3.14 x 10(-6) mol/l for nucleus caudate and 11.74 +/- 4.85 x 10(-6) mol/l for cerebral cortex). Therefore, it seems that transport system for adenosine plays the main role in tiazofurin brain uptake, but another transport mechanism is also involved in this process. Depletion of perfusing medium from sodium ions did not cause significant change in volume of distribution values for [3H]-tiazofurin in guinea pig cerebral cortex.

Tiazofurine uptake by the isolated guinea pig heart.

Tiazofurine is a selective inhibitor of the enzyme inosine monophosphate dehydrogenase, and exhibits potent antitumor activity. Considering the potential side effects on the heart, [3H] tiazofurine uptake into the cardiomyocytes, as well as the mechanism of transport, were studied in the isolated perfused guinea pig heart, using the rapid single circulation, paired-tracer technique. The maximal cellular uptake (Umax) of [3H] tiazofurine ranged from 19% to 25% of the injected dose, with total cellular uptake (Utot) ranging 12.1-15.6%. The addition of unlabeled tiazofurine caused inhibition of [3H] tiazofurine uptake, with a Umax value of 9.06 +/- 4.6%. Therefore, the uptake of tiazofurine into cardiomyocytes could be considered a saturable process. The inhibition of [3H] tiazofurine uptake caused by adenosine and dipyridamole was of the same degree as the inhibition by unlabeled tiazofurine. Thus, it can be assumed that nucleosides' transport system(s) are involved in transport of tiazofurine into myocardial cells.

Slow penetration of [3H] tiazofurin into guinea pig brain by a saturable mechanism.

The aim of this study was to investigate the transport of tiazofurin (2-beta-D-ribofuranosyl thiazole-4-carboxamide) across the blood-brain barrier (BBB) using brain vascular perfusion and capillary depletion method in the guinea pig. Values for volume of distribution of [3H] tiazofurin in brain increased slowly and almost linearly in time, from 1% of its plasma concentration at 1 min to 5% after 15 min of perfusion. Unidirectional transport constant Kin was 2.61 +/- 0.21 x 10(-3) ml/min/g. According to these results, it is evident that tiazofurin slowly penetrates the BBB. Addition of unlabeled tiazofurin to the perfusing medium caused a significant decrease in the uptake of [3H] labeled tiazofurin (Kin = 0.77 +/- 0.1 x 10(-3) ml/min/g). Therefore, penetration of tiazofurin from blood into brain seems to be a saturable process. Presence of potential inhibitors of tiazofurin blood-to-brain transport (adenosine and dipyridamole) did not cause complete inhibition of tiazofurin brain uptake. Thus, it could be assumed that transport of tiazofurin from blood into brain is only partially mediated by the nucleosides transport system. The results obtained using capillary depletion method show that tiazofurin that passes across the BBB is accumulated mostly in the brain tissue and not in brain capillaries endothelial cells.

Penetration of [3H] tiazofurin into guinea pig eye by a saturable mechanism.

This study investigated the transport of tiazofurin (2-beta-D-ribofuranosyl thiazole-4-carboxamide) across the blood-aqueous humor barrier, using the vascular perfusion method in the guinea pig. Volume of distribution (Vd) of [3H] tiazofurin increased almost linearly in time, from 4% of its plasma concentration at 3 min to 10% after 12 min of perfusion. Unidirectional transport constant, K(in) was 7.01 +/- 1.06 x 10(-3) ml/min/g. These results indicate that tiazofurin penetrates the aqueous humor to a considerable extent. Addition of unlabelled tiazofurin to the perfusing medium caused a significant decrease in the uptake of [3H] labelled tiazofurin (K(in) = 2.60 +/- 0.91 x 10(-3) ml/min/g). Therefore, penetration of tiazofurin from blood into aqueous humor seems to be a saturable process with a diffusional component that cannot be disregarded. Such findings could be of considerable importance since this molecule is known to affect tissue metabolism.