Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats.

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2.

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.

Blood-brain barrier produces significant efflux of L-aspartic acid but not D-aspartic acid: in vivo evidence using the brain efflux index method.

The brain efflux index method has been used to clarify the mechanism of efflux transport of acidic amino acids such as L-aspartic acid (L-Asp), L-glutamic acid (L-Glu), and D-aspartic acid (D-Asp) across the blood-brain barrier (BBB). About 85% of L-[3H]Asp and 40% of L-[3H]Glu was eliminated from the ipsilateral cerebrum within, respectively, 10 and 20 min of microinjection into the brain. The efflux rate constant of L-[3H]Asp and L-[3H]Glu was 0.207 and 0.0346 min(-1), respectively. However, D-[3H]Asp was not eliminated from brain over a 20-min period. The efflux of L-[3H]Asp and L-[3H]Glu was inhibited in the presence of excess unlabeled L-Asp and L-Glu, whereas D-Asp did not inhibit either form of efflux transport. Aspartic acid efflux across the BBB appears to be stereospecific. Using a combination of TLC and the bioimaging analysis, attempts were made to detect the metabolites of L-[3H]Asp and L-[3H]Glu in the ipsilateral cerebrum and jugular vein plasma following a microinjection into parietal cortex, area 2. Significant amounts of intact L-[3H]Asp and L-[3H]Glu were found in all samples examined, including jugular vein plasma, providing direct evidence that at least a part of the L-Asp and L-Glu in the brain interstitial fluid is transported across the BBB in the intact form. To compare the transport of acidic amino acids using brain parenchymal cells, brain slice uptake studies were performed. Although the slice-to-medium ratio of D-[3H]Asp was the highest, followed by L-[3H]Glu and L-[3H]Asp, the initial uptake rate did not differ for both L-[3H]Asp and D-[3H]Asp, suggesting that the uptake of aspartic acid in brain parenchymal cells is not stereospecific. These results provide evidence that the BBB may act as an efflux pump for L-Asp and L-Glu to reduce the brain interstitial fluid concentration and act as a static wall for D-Asp.

Association between early-onset alcoholism and the dopamine D2 receptor gene.

We examined the allelic association between the dopamine D2 receptor (DRD2) gene and alcoholism in 100 biologically unrelated Japanese alcoholics and 93 unrelated controls. Genomic DNA was prepared from peripheral white blood cells using the phenol-chloroform method. A 310-bp region surrounding the TaqA site at the DRD2 locus was amplified by polymerase chain reaction (PCR), and the PCR product was incubated with TaqI. The A1 allele remained intact while the A2 allele was cut. The frequency of the A1/A1 genotype and the frequency of the A1 allele were higher in early-onset alcoholics than in controls, P < 0.05 and P < 0.01, respectively. Moreover, the frequency of the A1/A1 genotype and the frequency of the A1 allele were higher in early-onset alcoholics with family histories of alcohol dependence than in controls, P < 0.01 and P < 0.01, respectively. The results indicate that the DRD2 gene is associated with susceptibility to early-onset alcoholism, and that each additional A1 allele shifts onset of alcoholism to an earlier age.

Effect of antipsychotic drugs on the gene expression of NMDA receptor subunits in rats.

It has been hypothesized that glutamatergic neurons play an important role in clinical manifestations of schizophrenia and that the therapeutic effect of antipsychotic drugs is related to glutamatergic neurotransmission. To elucidate the effect of antipsychotic drugs on glutamatergic transmission, we examined gene expressions of NMDA receptor subunits R1, R2A, R2B and R2C in the whole brains of rats after acute and chronic administrations of haloperidol and sulpiride, using the Northern blot technique. The levels of NMDAR2B mRNAs decreased after the acute administration of haloperidol, but showed no change after the chronic administration. The levels of NMDAR2A and R2B mRNAs decreased after the acute administration of sulpiride, whereas the levels of R2A and R2B increased following the chronic administration. Neither haloperidol nor sulpiride influenced NMDAR1 mRNA levels. These data support differential expression of NMDA receptor subunits in rats upon treatment with haloperidol and sulpiride. The results imply that NMDAR2 subunits may be crucial in the regulation and modification of antipsychotic drugs.

A chance SPECT study of ictal aphasia during simple partial seizures.

We report obtaining an ictal single photon emission computed tomographic (SPECT) scan in a right-handed 51-year-old man who had an astrocytoma in the left cerebral hemisphere and simple partial seizures characterized by aphasia. An epileptic seizure producing loss of speech and right-sided facial twitching occurred by chance during a SPECT scan. During the attack, he was unable to speak, but auditory comprehension and writing were intact. Ictal SPECT showed an area of increased perfusion in the left frontal cortex, with the area of highest perfusion involving the left frontal operculum to the inferior part of the left precentral gyrus. Interictal SPECT showed hypoperfusion in the same area. These SPECT findings suggest that the frontal operculum of the dominant hemisphere is one of the regions that can give rise to epileptic aphasia.

Paracentric inversion of chromosome 9 with schizoaffective disorder.

A 36-year-old man with schizoaffective disorder was found to have a de novo 46 XY inv (9) (q31.2q34.3). Phenotypical abnormalities are short stature, depressed nasal bridge, hypertelorism and slender shoulders. Paracentric inversion of chromosome 9 is a rare chromosome aberration. This case suggests a new candidate region related to schizoaffective disorder.

Positive association between a DNA sequence variant in the serotonin 2A receptor gene and schizophrenia.

Sixty-two patients with schizophrenia and 96 normal controls were investigated for genetic association with restriction fragment length polymorphisms (RFLPs) in the serotonin receptor genes. A positive association between the serotonin 2A receptor gene (HTR2A) and schizophrenia was found, but not between schizophrenia and the serotonin 1A receptor gene. The positive association we report here would suggest that the DNA region with susceptibility to schizophrenia lies in the HTR2A on the long arm of chromosomes 13.

Lack of point mutation of the APP gene in sporadic Alzheimer's disease in Japanese.

We investigated point mutations of the APP gene in 66 patients with sporadic Alzheimer's disease (AD) and 180 normal individuals by use of the PCR (polymerase chain reaction) method. Both the AD patients and the normal individuals were Japanese. We extracted DNA from blood samples using the phenol-chloroform method and amplified exons 16 and 17 of the APP gene by PCR. PCR products were digested by MBO-II (exon 16) and BCL-1(exon 17). Electrophoresis was carried out with 3% agarose gel and the separated fragments were stained with ethidium bromide. In addition we investigated other point mutations of exons 16 and 17 by use of the PCR-SSCP (single stranded conformation polymorphisms) method, and found no fragments that exhibited point mutations in the AD patients and normal individuals. These findings indicate that the presence of point mutation of the APP gene is not a major cause of AD in the Japanese population.

Neuroendocrine studies in dementia patients: responses of plasma GH and PRL following bromocriptine administration.

Bromocriptine stimulates growth hormone (GH) secretion at the hypothalamus and suppresses prolactin (PRL) secretion at the pituitary level. We administered bromocriptine to 30 patients with dementia of the Alzheimer type (DAT), 30 patients with multi-infarct dementia (MID) and 22 age matched healthy controls, and compared response patterns of GH and PRL. Incomplete PRL suppressive responses (suppression rate < 50%) were seen in 36.7% of DAT patients and in 30.0% of MID patients, indicating that both groups had the same degree of pituitary dysfunctions. Blunted GH responses (< 5 ng/ml) were seen in 93.3% of DAT patients, in 63.3% of MID patients and in 31.8% of the controls. The results indicate that neuroendocrine regulation of GH is more selectively and severely damaged in DAT patients than in MID patients at the hypothalamus.

Lack of association between bipolar affective disorder and tyrosine hydroxylase DNA marker.

Sixty-eight patients with bipolar affective disorder and 88 controls were investigated for genetic association of tyrosine hydroxylase (TH) restriction fragment length polymorphisms (RFLPs). No significant association between bipolar affective disorder and TH was found. Thus the hypothesis that TH is involved in the pathogenesis of bipolar affective disorder was not supported.

A clinical study of hypergraphia in epilepsy.

Fifteen patients with epilepsy and hypergraphia were compared with 32 patients with epilepsy but without hypergraphia. The number of previous psychiatric episodes, the number of Washington Psychosocial Seizure Inventory (WPSI) items indicating emotional maladjustment, and the number of CT scan abnormalities were significantly greater in the hypergraphic patients than in the non-hypergraphic patients. Cognitive performance, EEG laterality and the scores of WPSI items related to the psychological stress of seizures did not differ significantly between the two groups. Hypergraphia reflects changes in emotional responsiveness secondary to organic temporal lobe lesions.

P300 in first degree relatives of schizophrenics.

It has been reported that the amplitude of P300 is low in schizophrenic patients and high risk children. We recorded P300 components of the event-related potentials in first degree relatives of schizophrenic propositi who were considered to be over the age limit of the risk period for manifestation of schizophrenia and compared them with those of schizophrenic patients and controls. Both the first degree relatives and the schizophrenic patients showed lower P300 amplitude than the controls. There was no significant difference between the first degree relatives and schizophrenic patients in the P300 amplitude. These results would indicate that a low P300 amplitude is a trait marker for schizophrenia.

Failure to manipulate objects secondary to active touch disturbance.

A peculiar type of sensori-motor disturbance consequent to a lesion in the contralateral postcentral gyrus was reported. The symptom was characterized by motor clumsiness of the left hand without loss of strength and with preserved finger movements on visual imitation. Motor difficulty was most marked when the patient had to manipulate an object. The analysis of the patient's behavior and of his sensory deficits suggests that the basis of the clumsiness was a deficit in active touch perception.

Event-related potential (P300) in epilepsy.

The P300 component of auditory event-related potential was studied in 39 patients with temporal lobe epilepsy (TLE), 26 with idiopathic generalized epilepsy (IGE) and 28 controls. The age-corrected P300 latencies were significantly longer in TLE patients compared with those in IGE patients and controls. Neither the duration of epilepsy nor clinical manifestation was related to the P300 component in the same epileptic syndrome. The age-corrected P300 latencies recorded from Cz were significantly prolonged in TLE patients with bilateral temporal EEG foci compared with those with unilateral focus. The effects of anti-epileptic drugs on the P300 component were not significant. Our findings imply that prolonged P300 latency in TLE patients, especially in those with bilateral EEG foci is due to damage of the hippocampus, which is potentially an epileptogenic focus.

Event-related potentials (P300) in myotonic dystrophy.

The P300 component of the auditory event-related potential in 8 patients with myotonic dystrophy was studied and compared with that of 13 healthy controls. Abnormalities of P300 (prolongation of the latency and/or decrease of the amplitude) were observed in 6. These observations imply that the function of cognitive and information processing are impaired in myotonic dystrophy.

Motor learning in ideomotor apraxia.

Using Luria's motor sequence test, we studied the learning ability of 34 right-handed patients with unilateral hemisphere lesions. Patients with ideomotor apraxia needed 4 to 6 trials in the test, while the remainder needed only 1 or 2. Ideomotor apraxics also required more time to complete the test. The lesions of patients who failed to master this test were not always found in the frontal lobes. These results suggest that the ability to learn motor sequences is impaired in apraxic patients and that the left hemisphere of the brain plays the major role in learning a new motor sequence. Patients with ideomotor apraxia are impaired not only when performing previously learned motor tasks but also when learning a new motor task.