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.

Chronic amphetamine intoxication and the blood-brain barrier permeability to inert polar molecules studied in the vascularly perfused guinea pig brain.

The brain vascular perfusion method, with a multiple-time brain uptake analysis, has been employed to study the effects of chronic amphetamine intoxication on the kinetics of entry of 2 inert polar molecules, D-[14C]mannitol (mol.wt. 180) and [3H]polyethylene glycol (PEG, mol.wt. 4000) into the forebrain of the guinea pig. The unidirectional transfer constants, Kin, determined from graphic analysis 14 and 20 days after chronic amphetamine treatment (5 mg/kg daily, i.p.) showed a marked time-dependent progressive enhancement of transfer for both molecules. The kinetic features of this entry suggest the opening up of pathways through the blood-brain barrier (BBB) which allows mannitol and PEG to pass into the brain at rates which are irrespective of their molecular size and/or lipophilia and these changes cannot be attributed to simple mechanical factors such as hypertension. This opening of the BBB was associated with changes in behaviour (increased locomotor activity, stereotypy, hypervigilance, social withdrawal, and loss of weight) seen in 14- and 20-day amphetamine-treated animals. At 7 and 28 days after the withdrawal of the amphetamine treatment, the behavioural manifestations were absent, and the Kin values for both molecules were not significantly different from those measured in normal control animals which had been treated with placebo injections. The present results suggest a reversible dysfunction of the BBB as a consequence of the chronic amphetamine intoxication which correlates with the behavioural syndrome induced in the guinea pig.

Effects of sensory-motor cortical lesions on blood-brain permeability in guinea pigs.

Effects of sensory-motor cortical lesions on the function of the blood-brain barrier in distant brain areas are poorly understood. Therefore a brain vascular perfusion method has been used to measure simultaneously the kinetics of entry of two inert polar molecules, D-[14C]mannitol (MW 180) and [3H]polyethylene glycol (PEG; MW 4000), into the parietal cortex, hippocampus, and caudate nucleus in guinea pigs with ipsilateral and contralateral sensory-motor cortical lesions. The graphically determined cerebral capillary unidirectional constant, Kin, indicated a marked increase in blood-to-brain transport of both molecules in all regions studied, the changes being significantly higher after contralateral lesion. The mannitol/PEG cerebrovascular permeability constant ratio, Pman/PPEG, suggested the opening up of channels that permit a flow of fluid carrying substances either with respect to (2 days after ipsilateral lesion) or irrespective of their molecular size, depending on the time after lesion. Amphetamine treatment in the guinea pigs with sensory-motor lesions induced more pronounced blood-brain barrier permeability changes for both molecules in distant brain areas.

Dynamic interaction between caudate nucleus and hypothalamus.

Continuous low frequency stimulation of the caudate nucleus by constant parameters leads to the appearance of a characteristic "caudate spindle" as well as other "inhibitory" electrophysiological responses (single unit activity intracellular recordings) varying in duration and amplitude in the cerebral cortex. Computerized data processing of the responses (amplitude and duration of individual spindles, numbers of single unit blocking and sizes of EPSP and IPSP) indicated increments and decrements appearing in stable and characteristic sequences. The same low frequency caudate stimulation produces fast bursts in the course of continuous stimulation. The variations and dynamic patterns of inhibitory caudate influences upon the cortex seem to be in equilibrium with the excitatory influences from the hypothalamus representing a unique regulatory system.

Telencephalon cytoarchitecture in Serranus scriba.

Cytoarchitectural analysis of the telencephalon in Serranus scriba fish enabled us to differentiate a rather specialized morphological structure. Nerve cells identified in this structures cannot be classified following classical categories met in higher vertebrates neither be related to the isodendritic type of nonspecialized neurons. A centripetal orientation of dendrites and exons from all regions to the striatum is clearly expressed. Numerical prevalence of large cells is remarkable for similar structures in mammals in their prenatal and postnatal ontogenesis.

Effect of vitamin B6 on gamma-aminobutyric acid level in rat brain.

Vitamin B6 injected intraperitoneally into rats 400 mg/kg body weight, has produced a statistically significant decrease in GABA level concentrations in hippocampus and cerebellum. Cerebral cortex, caudate nucleus and thalamus have shown the decrease in GABA concentrations, but these changes were not statistically significant. No remarkable behavioural changes were noted under such circumstances. The possible functional meaning of these results is discussed in relation to the role of GABA distribution in different brain regions and development of convulsions.

Histochemical distribution of acid mucopolysaccharides during circadian rhythm in the cerebellum of Serranus scriba.

Histochemical analysis of AMPS (AB-staining materials) in Serranus scriba brain showed its irregular distribution in different brain regions. The highest AMPS content was observed in the optocoele and ependymal cells lining it, above the marginal telencephalon and optic tectum as well as the molecular cerebellum layer. Particularly significant AMPS concentrations were noted in Purkinje cell regions. The telencephalon and optic tectum showed AB-negative-staining materials. Following histochemical changes during a 24 h period, AMPS concentrations were shown to follow circadian variations, especially in the Purkinje cell region. The highest AMPS content was noted during the morning and the lowest during the evening hours. The above changes were discussed in the context of the role of AMPS in neural functions.