Milo N. Lipovac

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.

KINETICS OF ARGININE-VASOPRESSIN UPTAKE AT THE BLOOD-BRAIN BARRIER

Uptake of arginine-vasopressin, VP, at the luminal side of the blood-brain barrier (BBB) was studied by means of an in situ brain perfusion technique in the guinea-pig. Kinetic experiments revealed a saturable peptide influx into the parietal cortex, caudate nucleus and hippocampus with Km between 2.1 and 2.7 microM, and Vmax ranging from 4.9 to 5.6 pmol.min-1.g-1. The non-saturable component, Kd, was not significantly different from zero. Influx of VP into the brain was not altered by the presence of the peptide fragments: VP-(1-8), pressinoic acid and [pGlu4,Cyt6]VP-(4-9) at 4.5 microM, nor yet by the aminopeptidase inhibitor, bestatin (0.5 mM) and the L-amino acid transport system substrates, L-tyrosine and L-phenylalanine at 5 mM. At a perfusate concentration of 4.5 microM, the V1-vasopressinergic receptor antagonist, d(CH2)5[Tyr(Me)2]VP, reduced VP influx; regional Ki values, assuming that the observed inhibitions were purely competitive, ranged between 4.7 and 8.5 microM. It is concluded that there is an apparent cerebrovascular permeability to circulating VP due to the presence of a carrier-mediated transport system for the peptide located at the luminal side. The mechanism for VP BBB uptake exhibits no affinity for peptide fragments and large neutral amino acids, but requires reception of the intact molecule, which may be the same initial step for both the BBB VP transporter and the V1-receptor.

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.

A saturable mechanism for transport of immunoglobulin G across the blood-brain barrier of the guinea pig

The existence of an immunological blood-brain barrier to homologous blood-borne immunoglobulin G (IgG) was investigated in the guinea pig using a vascular brain perfusion technique in situ. Cerebrovascular unidirectional transfer constants (Kin) for 125I-labeled IgG (2.5 micrograms/ml) estimated from the multiple-time brain uptake data, ranged from 0.53 to 0.58 ml min-1 g-1 X 10(3) in the parietal cortex, hippocampus, and caudate nucleus, the transfer rate being some 10 times higher than that for [3H]dextran (MW 70,000). In the presence of 4 mg/ml unlabeled IgG, unidirectional blood to brain transfer of 125I-IgG was markedly inhibited. Immunohistochemical analysis of the brain tissue after vascular perfusion with unlabeled IgG revealed a distribution of the blood-borne immunoglobulin in the endothelial cells of microvessels and in the surrounding perivascular tissue. It is concluded that there is a specific transfer mechanism for IgG at the blood-brain barrier in the guinea pig, which is saturated at physiological plasma levels of IgG.

Metaphit, an isothiocyanate analog of PCP, induces audiogenic seizures in mice

Metaphit induces audiogenic seizures in mice. The most severe clonic/tonic seizures occur 18-24 h after metaphit administration. After 48 h the incidence of the seizure episodes begin to diminish. These audiogenic seizures can be prevented by the administration of either PCP or MK-801 24 h after metaphit and 30 min prior to audio stimulation. These seizures may be due to a modulation of the PCP recognition site by metaphit which results in an enhanced probability that the NMDA/PCP ion channels are open.

Saturable mechanism for delta sleep-inducing peptide (DSIP) at the blood-brain barrier of the vascularly perfused guinea pig brain

Cellular uptake of [125I] labelled DSIP at the luminal interface of the blood-brain barrier (BBB) was studied in the ipsilateral perfused in situ guinea pig forebrain. Regional unidirectional transfer constants (Kin) calculated from the multiple-time brain uptake analysis were 0.93, 1.33 and 1.66 microliter.min-1 g-1 for the parietal cortex, caudate nucleus and hippocampus, respectively. In the presence of 7 microM unlabelled DSIP the brain uptake of [125I]-DSIP (0.3 nM) was inhibited, the values of Kin being reduced to 0.23-0.38 microliter.min-1 g-1, values that were comparable with the Kin for mannitol. The rapidly equilibrating space of brain, measured from the intercept of the line describing brain uptake versus time on the brain uptake ordinate, Vi, was greater for [125I]-DSIP than for mannitol; in the presence of unlabelled DSIP this was reduced to that of mannitol, and it was suggested that the larger volume for [125I]-DSIP represented binding at specific sites on the brain capillary membrane. L-tryptophan, the N-terminal residue of DSIP, in concentrations of 7 microM and 1 mM, inhibited Kin without affecting Vi. A moderate inhibition of Kin was obtained by vasopressin ([Arg8]-VP), but only at a concentration as high as 0.2 mM. The results suggest the presence of a high affinity saturable mechanism for transport of DSIP across the blood-brain barrier, with subsequent uptake at brain sites that are highly sensitive to L-tryptophan, and may be modulated by [Arg8]-VP.

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.

Differential brain penetration of cerebroprotective drugs.

The use of cerebroprotective drugs has become a ubiquitous practice in neurosurgery, but our understanding of their pharmacokinetics and mode of action in the brain is limited. It has been speculated that beneficial therapeutic effects of cerebroprotective drugs may depend on their transport across the blood-brain barrier (BBB). To test this hypothesis, we studied two structurally different, but pharmacologically potent, cerebroprotective compounds, namely, a calcium-entry blocker, nimodipine, and a glucocorticoid, methyl-prednisolone. Clinical and experimental evidence suggests that nimodipine may act as a protective agent in ischemic brain damage secondary to cardiac arrest (1). Methyl-prednisolone is commonly used in the treatment of cerebral edema secondary to neoplasia (2, 3). In this study, an in situ vascular brain perfusion technique in guinea-pigs (4), and a capillary-depletion method (5) were used to determine pharmacokinetics of nimodipine and methyl-prednisolone at the BBB. The results described below are derived from our ongoing studies.

Blood-Brain Transport of Vasopressin

Arginine-vasopressin (AVP) is a hormone/neuropeptide that regulates a number of peripheral (e.g., antidiuresis, glycogenolysis) and central (e.g., memory, learning) functions (1). AVP is also involved in control of brain water metabolism (2), brain edema (3), cerebrospinal fluid (CSF) formation (4), and secretion of pituitary peptides (1). A potential therapeutic application of AVP is that of memory disorders caused by brain trauma, Alzheimer’s disease and senile dementia (5). Also V1-receptor antagonists may be helpful in prevention and therapy of brain edema (6). However, the lack of adequate experimental evidence has discouraged a wider clinical application of AVP and its neuroactive analogs.

Opening of the Blood—Brain Barrier to D-Mannitol Induced by Sensorimotor Cortical Lesions in the Anaesthetized Guinea-pig

We applied the brain vascular perfusion method to determine the kinetics of entry to inert polar molecular D-[3H]-mannitol into the parietal cortex, hippocampus and caudate nucleus in guinea-pigs with sensorimotor (SM) and occipital (Oc) cortical lesions. In the control group of animals, with Oc lesions, only a moderate increase of blood-brain (BBB) permeability to D-mannitol was found after the ipsilateral lesion and no permeability changes occurred after the contralateral lesion, which was expressed by graphically determined unidirectional transfer constant, Kin. In a separate series of experiments, in the guinea-pigs with SM cortical lesions, estimated Kin values indicated a marked increase of blood-to-brain transport of D-[3H)-mannitol in all brain regions studied, the changes being significantly higher after the contralateral lesion. Amphetamine pretreatment in the guinea-pigs with SM and Oc cortical lesions induced more pronounced BBB permeability changes for D-[3H]-mannitol in all brain areas studied.