Browsing by Author "Segal, Malcolm B. (7201773258)"

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. © 1989.

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. © 1989.

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.

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 permeability of the blood-cerebrospinal fluid (CSF) barrier to 3H-labelled thyrotropin-releasing hormone (TRH), was studied at the blood-tissue interface of the isolated perfused choroid plexus of the sheep, using a rapid (if< 30 s), single circulation paired-tracer dilution technique, in which d-[14C]mannitol serves as an extracellular marker. Arterio-venous loss of 14C radioactivity reflects the percentage of the d-mannitol dose that crosses the blood-CSF barrier using a non-specific pathway. This loss suggests that the choroidal epithelium is moderately leaky. Cellular uptake of TRH, estimated by directly comparing venous dilution profiles of [3H]TRH and d-[14C]mannitol was independent of this leakiness. The unidirectional transport of TRH could not be saturated with unlabelled TRH at a concentration as high as 10 mM, but was markedly reduced by 10 mM proline and by inhibitor of amidase and aminopeptidase activity, bacitracin (2 mM). Permeability of the blood-brain barrier to [3H]TRH was studied in the adult rat, employing the intracarotid injection technique of Oldendorf25 in which [14C]butanol served as an 'internal standard'. Brain-uptake of 3H radioactivity corrected for residual vascular space indicated a low extraction from the blood of TRH during a 15 s period of exposure to the peptide. Self-inhibition of [3H]TRH uptake by unlabelled TRH (10 mM) could not be demonstrated, but l-proline (10 mM) and bacitracin (2 mM) strongly inhibited this uptake. It is concluded that similar mechanisms at the blood-CSF and blood-brain barriers tend to prevent TRH penetration into the brain; namely the absence of significant transport systems or binding at the barrier and peptide degradation by the barrier or by plasma enzymes. However, in the presence of bacitracin, which prevents deamidation and hydrolysis of TRH, there is still an arterio-venous loss of 3H radioactivity during a single capillary passage through the choroid plexus of the same order as that of d-[14C]mannitol. It is suggested that there may be a passage of some intact TRH by a non-specific pathway through the choroid plexus into the CSF. This passage from blood via the CSF followed by diffusion in the extracellular space of nervous structures adjacent to the ventricles may represent a route of entry for TRH into the brain. © 1985.

The permeability of the blood-cerebrospinal fluid (CSF) barrier to 3H-labelled thyrotropin-releasing hormone (TRH), was studied at the blood-tissue interface of the isolated perfused choroid plexus of the sheep, using a rapid (if< 30 s), single circulation paired-tracer dilution technique, in which d-[14C]mannitol serves as an extracellular marker. Arterio-venous loss of 14C radioactivity reflects the percentage of the d-mannitol dose that crosses the blood-CSF barrier using a non-specific pathway. This loss suggests that the choroidal epithelium is moderately leaky. Cellular uptake of TRH, estimated by directly comparing venous dilution profiles of [3H]TRH and d-[14C]mannitol was independent of this leakiness. The unidirectional transport of TRH could not be saturated with unlabelled TRH at a concentration as high as 10 mM, but was markedly reduced by 10 mM proline and by inhibitor of amidase and aminopeptidase activity, bacitracin (2 mM). Permeability of the blood-brain barrier to [3H]TRH was studied in the adult rat, employing the intracarotid injection technique of Oldendorf25 in which [14C]butanol served as an 'internal standard'. Brain-uptake of 3H radioactivity corrected for residual vascular space indicated a low extraction from the blood of TRH during a 15 s period of exposure to the peptide. Self-inhibition of [3H]TRH uptake by unlabelled TRH (10 mM) could not be demonstrated, but l-proline (10 mM) and bacitracin (2 mM) strongly inhibited this uptake. It is concluded that similar mechanisms at the blood-CSF and blood-brain barriers tend to prevent TRH penetration into the brain; namely the absence of significant transport systems or binding at the barrier and peptide degradation by the barrier or by plasma enzymes. However, in the presence of bacitracin, which prevents deamidation and hydrolysis of TRH, there is still an arterio-venous loss of 3H radioactivity during a single capillary passage through the choroid plexus of the same order as that of d-[14C]mannitol. It is suggested that there may be a passage of some intact TRH by a non-specific pathway through the choroid plexus into the CSF. This passage from blood via the CSF followed by diffusion in the extracellular space of nervous structures adjacent to the ventricles may represent a route of entry for TRH into the brain. © 1985.

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 (≃ 35%) whereas that of thymidine was less (≃ 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 thyroidinc 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 [ 14 C]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 thorold plexus whereas for guanosine and thymidine, transport is of greater importance.

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 (≃ 35%) whereas that of thymidine was less (≃ 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 thyroidinc 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 [ 14 C]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 thorold plexus whereas for guanosine and thymidine, transport is of greater importance.

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.