Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia.

Binding studies with [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), a specific serotonin1A (5-HT1A) receptor agonist, were done on the autopsied brains from control subjects and from patients with chronic schizophrenia. All the patients and controls were of the Japanese race. In the controls, representative Scatchard plots for the specific [3H]8-OH-DPAT bindings in the prefrontal cortex and hippocampus revealed a single component of high affinity binding site (Kd value = 5.7 and 5.9 nM, Bmax value = 80.1 and 101.0 fmol/mg protein, respectively). The [3H]8-OH-DPAT bindings to the prefrontal cortex and hippocampus were potently inhibited by serotonin (IC50 = 6.3 x 10(-9) M) and 5-HT1A agonists (IC50 = 5.0 x 10(-9) - 2.3 x 10(-7) M), while other neurotransmitters, 5-HT2 and 5-HT3 related compounds did not inhibit the binding (IC50 greater than 10(-5) M). The bindings were decreased in the presence of 0.1mM GTP and 0.1mM GppNHp but not in the presence of 0.1mM GMP. In the prefrontal and temporal cortices of schizophrenics, there was a significant increase in the specific [3H]8-OH-DPAT binding, by 40% and 60%, respectively, with no change in the hippocampus, amygdala, cingulum, motor cortex, parietal or occipital cortex, as compared to findings in the controls. Scatchard analysis showed that this increased binding reflects changes in the number of sites but not in the affinity. The effect of 0.1mM GppNHp on the binding to prefrontal cortex was observed in both controls and schizophrenic patients. The bindings were significantly greater in the schizophrenic patients than in controls, in the presence of 0.1mM GppNHp. Our findings suggest that there are GTP-sensitive 5-HT1A sites in the human brain and that selective increases in GTP-sensitive 5-HT1A sites in the prefrontal and temporal cortices of schizophrenics relate to the pathophysiology of schizophrenia.     

Phosphoinositide signalling system in platelets of schizophrenic patients and the effect of neuroleptic therapy.

Alterations in the phosphoinositide signalling system have been proposed as a possible biological marker of schizophrenia. We studied the levels of inositol 1,4,5-trisphosphate (IP3), cytosolic Ca2+ concentrations ([Ca2+]i), and the incorporation of [32P]-orthophosphate into inositol phospholipids and phosphatidic acid (PA) in blood platelets of neuroleptic-treated schizophrenics in comparison with controls. The [Ca2+]i was significantly higher in platelets of one month neuroleptic-treated patients (155+/-5.8 nM) in comparison with controls (95+/-5.4 nM). Neuroleptic therapy decreased the [Ca2+]i, but even after long-term therapy it remained significantly higher (114+/-5.7 nM) than in controls. Differences were also found in the level of IP3 between controls (30+/-4.0 pmol/10(9) platelets), drug-free schizophrenics (52+/-9.0 pmol/10(9) platelets) and treated patients (50+/-6.0 pmol/10(9) platelets). The increased turnover of PA was observed in platelets of neuroleptic-treated schizophrenic patients. The study suggests that the regulation of calcium homeostasis and pathways involved in the phosphoinositide signalling system are altered in the platelets of schizophrenics. Neuroleptic therapy did not remove the observed changes in [Ca2+]i and IP3 levels.     

Increased muscarinic cholinergic receptors in prefrontal cortices of medicated schizophrenics

Alterations in 3H-quinuclidinyl benzilate binding sites associated with muscarinic cholinergic receptors were investigated in orbito-frontal and medial frontal cortices from 12 schizophrenics, 6 on-drug and 6 off-drug cases, and from 10 controls. Significantly lower affinities of the sites were found in both areas of schizophrenics than controls. An increase in receptor number was shown only in the orbito-frontal cortex from schizophrenics. On-drug group of schizophrenics did, however, show a significant increase in receptor number and a significant decrease in affinity in both areas, while there were no significant differences in any binding parameters of off-drug schizophrenics from controls. Also in the caudate the similar results were obtained. It is, thus, concluded that alterations in muscarinic cholinergic receptors of schizophrenic patients result from long-term medication with antimuscarinic actions.     

Peripheral benzodiazepine binding sites in platelets of patients affected by mitochondrial diseases and large scale mitochondrial DNA rearrangements

BACKGROUND: The peripheral-type benzodiazepine receptors (PBR) are localized on the outer mitochondrial membrane, as a constituent of mitochondrial permeability transition (MPT)-pore. Among its hypothesized functions, the regulation of the mitochondrial respiratory chain and apoptosis have been suggested; in addition alterations of PBR site density have been shown in some neuropathologic conditions with putative mitochondrial involvement. The aim of this work has been to evaluate PBR kinetic binding parameters in platelets from patients affected by mitochondrial disorders (MD) with large-scale mitochondrial DNA deletions and reduced cytochrome c oxidase activity. MATERIALS AND METHODS: Using the specific PBR radioligand [(3) H] PK 11195, the kinetic binding parameters of PBR sites were determined in platelet membrane of 15 healthy subjects and 11 patients affected by different form of MD. RESULTS: Significant changes of dissociation constant (K(d)) and maximal number of binding sites (B(max)) values were evidenced in platelets of patients versus controls. In all patients the B(max) values were decreased (2,387.0 +/- 305.6 fmol/ mg proteins versus 4889.0 +/- 357.8 fmol/mg proteins, p< 0.05), whereas the K(d) values were higher in patients than controls (13.18 +/- 2.06 nM versus 5.63 +/- 0.46 nM, p< 0.05). CONCLUSIONS: These data suggest that the kinetic binding parameters of PBR are altered in MD and that the observed changes might be related to the mitochondrial dysfunction associated with MD.     

Platelet prostaglandin E1 hyposensitivity in schizophrenia: reduction of prostaglandin E1- or forskolin-stimulated cyclic AMP response in platelets

Cyclic 3',5'-adenosine monophosphate (cAMP) formation via prostaglandin E1 (PGE1)-or forskolin-stimulation were determined in washed intact platelets from 32 schizophrenic patients and 30 normal controls. Regarding basal cAMP levels in the platelets, there were no differences between schizophrenic patients and normal controls. Both PGE1-and forskolin-stimulated cAMP response reduced in platelets from schizophrenics compared with normal controls. These results suggested that platelets in schizophrenics were impaired not only in the adenylate cyclase unit per se but also extensively in the cAMP generating system coupled to a PGE1 receptor.     

Labelling of "Peripheral-Type" Benzodiazepine Binding Sites in the Rat Brain By Using [3H]PK 11195, an Isoquinoline Carboxamide Derivative: Kinetic Studies and Autoradiographic Localization

PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide] is a new ligand for the "peripheral-type" benzodiazepine binding sites, chemically unrelated to benzodiazepines. It displaces with a very high potency (IC50 congruent to 10(-9) M) [3H]-RO5-4864 (a benzodiazepine which specifically labels the peripheral-type sites) from its binding sites. [3H]PK 11195 binds to a membrane fraction from rat brain cortex and rat olfactory bulb in a saturable and reversible manner with a very high affinity (KD = 10(-9) M). The number of maximal binding sites was ten times greater in the olfactory bulb than in the brain cortex. The order of potency of several compounds as displacers at 25 degrees C (PK 11195 greater than RO5-4864 greater than diazepam greater than dipyridamole greater than clonazepam) demonstrates that [3H]PK 11195 binds to the peripheral-type benzodiazepine binding sites. The KD value for the [3H]PK 11195 binding is not affected by temperature changes, whereas RO5-4864 and diazepam affinities decrease with increasing temperatures. Autoradiographic images of [3H]PK 11195 binding to rat brain sections show that binding sites are mainly localized in the olfactory bulb, median eminence, choroid plexus, and ependyma. This ligand could be a useful tool to elucidate the physiological and pharmacological relevance of these binding sites.     

Schizophrenia and reduced cyclic AMP production: evidence for the role of receptor-linked events

Reduced cyclic AMP (cAMP) production has been found in platelets of schizophrenic patients. cAMP is generated physiologically as a result of a series of steps beginning with receptor activation by a ligand, progressing through activation of the enzyme protein, adenylate cyclase. The deficit of cAMP found in the schizophrenic population may occur at any one, or at multiple steps in this cascade. The present study attempts to discriminate whether impaired adenylate cyclase itself was responsible for the cAMP deficit or whether abnormalities in receptor events or linkage are present in schizophrenics. The production of cAMP following direct stimulation of adenylate cyclase by NaF was contrasted with receptor mediated activation of adenylate cyclase by prostaglandin E1 (PGE1) in disrupted platelet preparations from schizophrenics and normal controls. cAMP formation stimulated by NaF was not different in platelets of schizophrenics as compared to controls, however, platelets of schizophrenics showed reduced response to PGE1 stimulation. The authors interpret these findings as evidence for a membrane associated abnormality of either receptor or receptor-adenylate cyclase linkage in the schizophrenias.     

Increased serotonin2 (5-HT2) receptor binding as measured by 3H-lysergic acid diethylamide (3H-LSD) in the blood platelets of depressed patients

3H-Lysergic acid diethylamide (3H-LSD) binding, a putative measure of 5-HT2 receptor binding, was studied in the blood platelets of 29 depressed patients and 24 normal controls. The Bmax (maximum number of 3H-LSD binding sites) in the blood platelets of depressed patients was significantly greater than that of normal volunteers. This increase in Bmax was due to an increase in female depressed patients only. Bmax was significantly lower in female compared to male normal controls but there was no difference between male and female depressed patients. There was also no difference in Kd (an inverse measure of affinity of 3H-LSD binding to its sites) between normal controls and depressed patients. The correlations between Bmax of 3H-LSD binding and the Bmax of the 3H-imipramine binding site or the Vmax of 5-HT uptake sites were not significant. The role of serotonergic processes in the psychobiology of depression is discussed.     

Peripheral-type benzodiazepine receptors in human blood cells of patients affected by migraine without aura

The kinetic parameters at equilibrium of peripheral benzodiazepine receptors in platelets, lymphocytes and granulocytes of 15 patients affected by migraine without aura were tested using [3H]PK 11195, a specific radioligand for this receptor and compared with the same number of healthy controls: a statistically significant increase (platelets 212%, lymphocytes 203%, granulocytes 171%, as absolute percentage) in the maximal number of binding sites (B(max)) in all three patient samples, compared with healthy controls was detected; on the contrary, the values of the dissociation constant (K(d)) at equilibrium do not show any statistically significant variations between the two groups. These data further confirm the presence of peripheral biochemical alterations in migraine without aura. As peripheral benzodiazepine receptors appear to be involved in the regulation of the mitochondrial respiratory chain, the observed increase in B(max) might be related to the mitochondrial anomalies found in migraine disorders.     

3H]muscimol binding sites increased in autopsied brains of chronic schizophrenics

[3H]muscimol binding and glutamic acid decarboxylase (GAD) activity in the prefrontal cortex and caudate nucleus of autopsied brains from 19 chronic schizophrenics and 17 control subjects were investigated. In the schizophrenics, saturation analysis with varying concentrations of [3H]muscimol revealed an increase in the number of GABAA receptors, but there was no significant difference in the affinity. In addition, the enhancement of [3H]muscimol binding by diazepam was significantly greater in schizophrenics than in controls. GAD activity did not differ between controls and schizophrenics. The possibility that GABAergic mechanisms might play a role in case of chronic schizophrenia should be given further attention.     

Examination stress, platelet peripheral benzodiazepine binding sites, and plasma hormone levels

The maximal binding capacity (Bmax) and the equilibrium dissociation constant (KD) values for [3H]PK 11195 binding to peripheral benzodiazepine binding sites were measured in the platelets of subjects immediately after examination stress and 10 days later, as well as in unstressed controls. Increased (52%; p less than 0.05) Bmax, but unaltered KD, values were observed immediately after the stress as compared to the controls. Ten days after the stress, a slight decrease (15%) in the number of peripheral benzodiazepine binding sites was observed, but their values remained higher (29%) in comparison to unstressed controls. Levels of plasma stress hormones (cortisol, growth hormone, and prolactin) did not differ in the stressed subjects from those of the controls.     

Increased turnover of platelet phosphatidylinositol in schizophrenia.

The potential role of receptor-stimulated phosphatidylinositol (PI) hydrolysis in a signal transduction mechanism has been increasingly recognized. Earlier studies have suggested a defect in alpha-adrenergic receptor function in the platelets of schizophrenic patients. Little is known, however, about the mechanisms for PI synthesis, breakdown, and regulation in schizophrenia. The present study was undertaken to investigate the metabolic turnover of inositol phospholipids and inositol phosphates by incorporation of [3H]myoinositol or [32P]orthophosphate into resting and activated platelets of normal controls and schizophrenic patients with and without neuroleptic treatment. After 5 h incubation at 37 degrees C, the majority of [3H]myoinositol was incorporated into platelet PI. Following thrombin-induced platelet activation, there was rapid formation of 3H-labeled inositol phosphates (IPs) with inositol monophosphate (IP1) being the most abundant product. The thrombin-induced formation of platelet IPs was found significantly higher in both haloperidol-stabilized and drug-free schizophrenics than in normal control subjects. When platelets were prelabeled with [32P]orthophosphates, thrombin-induced formation of phosphatidic acid (PA) was also significantly higher in haloperidol-stabilized schizophrenics than in normal controls. It is thought that thrombin-induced platelet activation is mediated through hydrolysis of polyphosphoinositides (poly-PI). The present data thus may reflect an increased signal transduction in schizophrenia, which is mediated through neuroleptic-regulated inositol phospholipid hydrolysis.     

Platelet peripheral benzodiazepine receptors are decreased in Parkinson's disease.

Peripheral benzodiazepine (BDZ) receptors are located in a variety of tissues, including platelets, in the nuclear and/or mitochondrial membranes. We studied the density of peripheral BDZ receptors in platelets of 10 de novo Parkinson's disease (PD) patients, 18 PD patients treated with a levodopa/carbidopa combination, and in 15 healthy subjects matched for sex and age. The binding assay was conducted using [3H]PK 11195, a specific ligand for peripheral BDZ receptors. A significant decrease in the density of [3H]PK 11195 binding sites has been observed in PD patients with respect to controls (p less than 0.01), but not between de novo and treated PD patients. No correlation has been found between the decrease in density of [3H]PK 11195 binding sites in platelets and either the duration or severity of PD. Peripheral BDZ receptors are implicated in the regulation of mitochondrial respiratory function. Thus, their decrease in PD might parallel the abnormalities in mitochondrial function recently found in this neurologic disease.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Frontal Cortical and Left Temporal Glutamatergic Dysfunction in Schizophrenia

Glutamatergic mechanisms have been investigated in postmortem brain samples from schizophrenics and controls. D-[3H]Aspartate binding to glutamate uptake sites was used as a marker for glutamatergic neurones, and [3H]kainate binding for a subclass of postsynaptic glutamate receptors. There were highly significant increases in the binding of both ligands to membranes from orbital frontal cortex on both the left and right sides of schizophrenic brains. The changes are unlikely to be due to antemortem neuroleptic drug treatment, because no similar changes were recorded in other areas. A predicted left-sided reduction in D-[3H]aspartate binding was refuted at 5% probability, but not at 10%. Previously reported high concentrations of dopamine in left amygdala were strongly associated with low concentrations of D-[3H]aspartate binding in left polar temporal cortex in the schizophrenics. The findings are compatible with an overabundant glutamatergic innervation of orbital frontal cortex in schizophrenia. The results also suggest that schizophrenia may involve left-sided abnormalities in the relationship between temporal glutamatergic and dopaminergic projections to amygdala.     

P300 topographic asymmetries are present in unmedicated schizophrenics

Our laboratory has repeatedly found a left < right auditory P300 temporal lobe topographic asymmetry in right-handed, medicated schizophrenics. To determine whether this asymmetry was attributable to the effects of antipsychotic medications, we collected auditory “odd-ball” P300 event-related potentials from 14 right-handed, unmediated schizophrenics (withdrawn from medication for an average of 21 days) and 14 right-handed, normal controls. Analysis of normalized P300 amplitudes showed a statistically significant difference in the voltage distributions between groups (a group by temporal electrode site interaction) that was consistent with a left < right temporal voltage asymmetry in schizophrenics but not in the normal controls. We conclude that P300 topographic asymmetries are present in unmedicated schizophrenics. These data are compatible with the growing body of data suggsting left temporal lobe structural abnormalities in schizophrenia.     

Modulatory Effects of Thyroxine Treatment on Central and Peripheral Benzodiazepine Receptors in the Rat

The effects of 10 days of D-thyroxine (T4) treatment on central benzodiazepine (BZ) receptors in the brain and on peripheral-type BZ binding sites in the heart, kidney, and testis of rats were studied. The experimental hyperthyroidism resulted in an increase in the density of cortical central BZ receptors, without any alteration of the affinity of the receptors to [3H]flunitrazepam. The increase in cortical central BZ receptors was also accompanied by the up-regulation of peripheral BZ binding sites in the heart, kidney, and testis. The affinity of the peripheral BZ binding sites for the ligand [3H]PK 11195 was not affected by T4 treatment in any of these three organs. The increase in the density of brain cortical central BZ receptors was less prominent than the increase in the peripheral BZ binding sites. The modulatory effect of T4 treatment on central and peripheral BZ receptors might be attributed to the direct interaction of the thyroid hormone at these sites or might reflect a physiological compensatory adaptation mechanism to thyrotoxicosis associated with hypermetabolism, anxiety, and stress.     

Portacaval anastomosis causes selective alterations of peripheral-type benzodiazepine receptor expression in rat brain and peripheral tissues.

There is a growing body of evidence to suggest that peripheral-type benzodiazepine receptors (PTBRs) and their endogenous ligands are implicated in the pathogenesis of end-organ failure in chronic liver disease. Portal-systemic encephalopathy, a major neuropsychiatric complication associated with chronic liver disease, results in activation of brain PTBR and probably in peripheral organs. In order to address these issues, PTBR mRNA was measured using semi-quantitative RT-PCR in extracts of cerebral cortex, kidney and testis of rats four weeks after end-to-side portacaval anastomosis and sham-operation (controls). Densities of PTBR sites were measured concomitantly by in vitro receptor binding using the selective PTBR ligand [3H]PK11195. Portacaval shunting resulted in a 2 to 3-fold increase in expression of PTBR in brain and kidney and a 37% reduction in expression in testis. Densities of [3H]PK11195 sites changed in parallel with the alterations of gene expression. These findings suggest that selective alterations of PTBR expression are implicated in the pathogenesis of peripheral tissue hypertrophy (kidney) and/or atrophy (testis) which accompanies portal-systemic shunting in chronic liver failure. In brain, activation of PTBR could result in an increase in the production of neurosteroids with potent inhibitory action in the CNS, which could contribute to the pathogenesis of portal-systemic encephalopathy.     

A subpopulation of platelets responds to thrombin- or SFLLRN-stimulation with binding sites for factor IXa

Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500-600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin- or SFLLRN-activated platelets. Concentrations of these agonists that are half-maximal or maximal in kinetic studies resulted in only a small subpopulation (4-20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 +/- 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (approximately 10%) of PAR 1-stimulated platelets expose approximately 6000 factor IXa binding sites/platelet.