Browsing by Author "Redzic, Zoran B. (6602453895)"

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 (K(i) and K(d)a), 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 K(m) 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 (K(i) = 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.

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 (K(i) and K(d)a), 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 K(m) 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 (K(i) = 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.

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (Umax) 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 Umax). 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 RCSF/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. © 2001 Elsevier Science B.V.

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (Umax) 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 Umax). 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 RCSF/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. © 2001 Elsevier Science B.V.

The brain efflux of radiolabelled hypoxanthine in the rat was rapid in the first minute after injection [Keff(i)=0.21±0.06 min-1], which was saturable with a Vmax=13.08±0.81 nM min-1 g-1, and a high Km.app (67.2±13.4 μM); the Ki.app for inosine was 31.5±7.6 μM. 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. © 2001 Elsevier Science B.V.

The brain efflux of radiolabelled hypoxanthine in the rat was rapid in the first minute after injection [Keff(i)=0.21±0.06 min-1], which was saturable with a Vmax=13.08±0.81 nM min-1 g-1, and a high Km.app (67.2±13.4 μM); the Ki.app for inosine was 31.5±7.6 μM. 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. © 2001 Elsevier Science B.V.

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na + -independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63±3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of K m (20.72±2.42 μM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61±2.28 μM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo. © 2002 Elsevier Science B.V. All rights reserved.

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na + -independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63±3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of K m (20.72±2.42 μM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61±2.28 μM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo. © 2002 Elsevier Science B.V. All rights reserved.