Ljubisa Rakić

Transport of Leucine‐Enkephalin Across the Blood‐Brain Barrier in the Perfused Guinea Pig Brain

Abstract: Transport of [tyrosyl‐3, 5−3H]enkephaIin‐(5‐L‐leucine) ([3H]Leu‐enkephalin) across the blood‐brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kim) estimated from the multiple‐time uptake data for [3H]Leu‐enkephalin ranged from 3.62 ± 10−3 to 3.63 ± 10−3 ml min−1 g−1in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu‐enkephalin was not inhibited by unlabelled L‐tyrosine (the N‐terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood‐brain barrier. By contrast, 2 mM unlabelled Leu‐enkephalin strongly inhibited the unidirectional blood‐to‐brain transport of [3H]Leu‐enkephalin by 74‐78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl‐glycyl‐glycyl‐phenylalanine (without the C‐terminal leucine of Leu‐enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl‐glycyl‐phenylalanyl‐leucine (without the N‐terminal tyrosine) at 5 mM was without effect on Leu‐enkephalin transport. Unidirectional brain uptake of Leu‐enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM(I mg/ml), suggesting that there is no binding of Leu‐enkephalin to opioid receptors at the blood‐brain barrier. It is concluded that there is a specific transport mechanism for Leu‐enkephalin at the blood‐brain barrier in the guinea pig.

Slow Penetration of Thyrotropin-Releasing Hormone Across the Blood-Brain Barrier of an In Situ Perfused Guinea Pig Brain

Transport of 3H‐labelled thyrotropin‐releasing hormone (TRH) across the blood‐brain barrier was studied in the ipsilateral perfused in situ guinea pig forebrain. The unidirectional transfer constant (Kin) calculated from the multiple time brain uptake analysis ranged from 1.14 × 10‐−3 to 1.22 × 10‐−3 ml min−1 g−1, in the parietal cortex, caudate nucleus, and hippocampus. Regional Kin values for [3H]TRH were significantly reduced by 43–48% in the presence of an aminopeptidase and amidase inhibitor, 2 mM bacitracin, suggesting an enzymatic degradation of tripeptide during interaction with the blood‐brain barrier. In the presence of unlabelled 1 mM TRH and 2 mM bacitracin together, a reduction of [3H]TRH regional Kin values similar to that obtained with 2 mM bacitracin alone was obtained. l‐Prolinamide, the N‐terminal residue of tripeptide, at a 10 mM level had no effect on the kinetics of entry of [3H]TRH into the brain. The data indicate an absence of a specific saturable transport mechanism for TRH presented to the luminal side of the blood‐brain barrier. It is concluded that intact TRH molecule may slowly penetrate the blood‐brain barrier, the rate of transfer being some three times higher than that of d‐mannitol.

An introduction to the blood-brain barrier

History of basic concepts transport of glucose and amino-acids in the central nervous system peptides and proteins transport of some precursors of nucleotides and some vitamins experimental models in the study of pathology of the blood-brain barrier.

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep

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 Km (20.72+/-2.42 microM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61+/-2.28 microM) 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.

Sulfinosine-induced cell growth inhibition and apoptosis in human lung carcinomas in vitro

In spite of tremendous effort for improvedtherapy, lung cancer remains the leadingcause of cancer-related deaths worldwide.In the present study, we used the novelpurine ribunocleoside sulfinosine andevaluated its antiproliferative andapoptotic outcome on the non-small celllung carcinoma cell line (NSCLC) and thesmall cell lung carcinoma cell line (SCLC).Using a BrdU incorporation-test sulfinosineinhibited cell growth in a dosedependent-manner. ID50 values were4.65 ± 0.17 μM in the case of NSCLCcells, and 3.59 ± 0.81 μM in thecase of SCLC cells. MTT testing revealedthat IC50 values were 6.24 ±0.77 μM for NSCLC and 5.68 ±0.58 μM for SCLC. Inhibitoryconcentrations (IC50 and ID50)for sulfinosine were nonsignificantly lowerin SCLC cells compared to NSCLC cells,indicating similar susceptibility of thecells. Flow-cytometric analysis, TUNELstaining, DNA laddering and cell deathELISA test were used to investigateapoptotic cell death. Our resultsdemonstrated that high concentrations ofsulfinosine can cause typical DNAladdering, a hallmark for apoptosis.Evidence of free nucleosomes and enzymaticlabeling of fragmented DNA confirmedapoptosis involvement in sulfinosinecytotoxicity. In addition, flow-cytometricanalysis showed that 25 μM sulfinosinearrested cell cycle progression atthe G2M phase and induction ofapoptosis in both cell lines. From theseresults, we concluded that sulfinosine mayact as an anticancer agent and furtherstudies may prove its efficacy in lungcancer cells. Thus the biological effectsof sulfinosine may be due to modulation ofcell growth, cell death, and cell cycleregulatory molecules.

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.

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.

History and Basic Concepts

The concept of the blood-brain barrier derives from the classical studies of the pioneers in chemotherapy, such as Ehrlich, who administered dyestuffs parenterally in the hope that they would attack infective organisms. Thus Ehrlich observed that many dyes, after intravenous injection, stained the tissues of practically the whole body, while the brain was spared. Later, Lewandowsky (1900) showed that the Prussian blue reagents (iron salt and potassium ferrocyanide) did not pass from blood to brain, and he formulated clearly the concept of the blood-brain barrier (Bluthirnschranke). The more definitive demonstration of the barrier we owe to Goldmann, who showed (1909) that, after intravenous injection with trypan blue, the brain was unstained; the dye did not enter the cerebrospinal fluid (CSF), although the choroid plexuses and meninges were stained. In a second paper (Goldmann, 1913), he described experiments in which trypan blue was injected into the CSF; in this event, the brain tissue was strongly stained, so that Goldmann rightly concluded that there was, indeed, a barrier between blood, on the one hand, and brain tissue on the other. Any argument that the failure to stain the brain with trypan blue after intravenous injection was due to a peculiar staining feature of the nervous tissue was negated by this fundamental ‘second experiment’, the first experiment being the demonstration that nervous tissue was unstained after intravenous injection.

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 effects of NO synthesis inhibition on the uptake of endogenous nucleosides into the rat brain

Brain uptake of 3H endogenous nucleosides in rats was measured by Brain Uptake Index method, using 14C-sucrose as a referent molecule. The values obtained in control group were 4.68±0.93 for adenosine, 4.10±0.72 for guanosine and 1.14±0.72 for thymidine, indicating significant blood-to-brain transport of purine nucleosides. NO-synthase inhibition was induced by i.p. application of 25 mg/kg 1-ω-nitro-arginin methyl ester (L-NAME) and 5mg/kg diphenylethiodonium (brain parenchyma NOS inhibitor). HPLC analysis showed that the concentration of L-NAME in brain was sufficient to cause inhibition of NOS. Application of L-NAME resulted in significant inhibition of brain uptake for both purine nucleosides. Unlike adenosine, in the case of guanosine a correlation between the rate of brain uptake inhibition and the L-NAME concentration in plasma, but not in brain, was obtained. Application of diphenylethiodonium also caused a significant decrease in both guanosine and adenosine brain uptake. The obtained results were not significantly different from the BUI values obtained after preapllication of L-NAME. Our results suggest that NOS inhibition decreases brain uptake of purine nucleosides. Such effect(s) are probably due to the inhibition of brain parenchyma NOS.