Reduced connectivity of the auditory cortex in patients with auditory hallucinations: a resting state functional magnetic resonance imaging study.

BACKGROUND: Previous research has reported auditory processing deficits that are specific to schizophrenia patients with a history of auditory hallucinations (AH). One explanation for these findings is that there are abnormalities in the interhemispheric connectivity of auditory cortex pathways in AH patients; as yet this explanation has not been experimentally investigated. We assessed the interhemispheric connectivity of both primary (A1) and secondary (A2) auditory cortices in n=13 AH patients, n=13 schizophrenia patients without auditory hallucinations (non-AH) and n=16 healthy controls using functional connectivity measures from functional magnetic resonance imaging (fMRI) data. METHOD: Functional connectivity was estimated from resting state fMRI data using regions of interest defined for each participant based on functional activation maps in response to passive listening to words. Additionally, stimulus-induced responses were regressed out of the stimulus data and the functional connectivity was estimated for the same regions to investigate the reliability of the estimates. RESULTS: AH patients had significantly reduced interhemispheric connectivity in both A1 and A2 when compared with non-AH patients and healthy controls. The latter two groups did not show any differences in functional connectivity. Further, this pattern of findings was similar across the two datasets, indicating the reliability of our estimates. CONCLUSIONS: These data have identified a trait deficit specific to AH patients. Since this deficit was characterized within both A1 and A2 it is expected to result in the disruption of multiple auditory functions, for example, the integration of basic auditory information between hemispheres (via A1) and higher-order language processing abilities (via A2).

Emotional prosodic processing in auditory hallucinations.

Deficits in emotional prosodic processing, the expression of emotions in voice, have been widely reported in patients with schizophrenia, not only in comprehending emotional prosody but also expressing it. Given that prosodic cues are important in memory for voice and speaker identity, Cutting has proposed that prosodic deficits may contribute to the misattribution that appears to occur in auditory hallucinations in psychosis. The present study compared hallucinating patients with schizophrenia, non-hallucinating patients and normal controls on an emotional prosodic processing task. It was hypothesised that hallucinators would demonstrate greater deficits in emotional prosodic processing than non-hallucinators and normal controls. Participants were 67 patients with a diagnosis of schizophrenia or schizoaffective disorder (hallucinating=38, non-hallucinating=29) and 31 normal controls. The prosodic processing task used in this study comprised a series of semantically neutral sentences expressed in happy, sad and neutral voices which were rated on a 7-point Likert scale from sad (-3) through neutral (0) to happy (+3). Significant deficits in the prosodic processing tasks were found in hallucinating patients compared to non-hallucinating patients and normal controls. No significant differences were observed between non-hallucinating patients and normal controls. In the present study, patients experiencing auditory hallucinations were not as successful in recognising and using prosodic cues as the non-hallucinating patients. These results are consistent with Cutting's hypothesis, that prosodic dysfunction may mediate the misattribution of auditory hallucinations.

Competence to give informed consent in acute psychosis is associated with symptoms rather than diagnosis.

To investigate the association between competence to give informed consent to treatment, specific symptomology and diagnostic category, 110 inpatients diagnosed with DSM-IV acute schizophrenia (n = 64), schizoaffective disorder (n = 25) and bipolar affective disorder (n = 21) were interviewed using the MacArthur Competence Assessment Tool for Treatment (MacCAT-T) and the Positive and Negative Syndrome Scale (PANSS). Results indicated no significant difference in competence between the three disorders. Elevated positive, cognitive and excitement PANSS factor scores had lower MacCAT-T scores. Further analyses indicated symptoms that impair cognition; particularly, conceptual disorganisation and poor attention were most consistently related to poor performance on competence tests.

Hippocampal volume in first-episode psychoses and chronic schizophrenia: a high-resolution magnetic resonance imaging study.

BACKGROUND: It has been proposed that the hippocampus is a potential site for a neurodevelopmental lesion in schizophrenia. While smaller hippocampal volumes have been described in chronic schizophrenia, there have been few magnetic resonance imaging studies in first-episode psychosis. Furthermore, no studies have examined the specificity of this finding to first-episode schizophrenia, compared with first-episode affective psychosis. METHODS: Hippocampal and whole-brain volumes were estimated using high-resolution magnetic resonance imaging in 140 controls, 46 patients with chronic schizophrenia, and 32 patients with first-episode psychosis. RESULTS: Patients with chronic schizophrenia and first-episode psychosis had significantly smaller hippocampal volumes as compared with controls. Within the first-episode group, both patients with schizophrenia/schizophreniform psychosis and those with affective psychosis had smaller left hippocampal volumes as compared with controls. Smaller right hippocampal volumes were associated with age and illness duration in patients with chronic schizophrenia. Hippocampal volumes were not correlated with age of illness onset or medication dosage in either patient group. CONCLUSIONS: These data show that smaller hippocampal volumes are present from the onset of illness. While these findings would support the neurodevelopmental model of schizophrenia, the finding of smaller left hippocampal volume in patients with first-episode schizophrenia and affective psychosis does not support the prediction that smaller hippocampi are specific to schizophrenia. The association of smaller right hippocampal volumes with increased illness duration in chronic schizophrenia suggests either that there is further neurodegeneration after illness onset or that bilateral small hippocampi predict chronicity.

D5 dopamine receptors mediate estrogen-induced stimulation of hypothalamic atrial natriuretic factor neurons.

Whereas progesterone and dopamine share a common central pathway to modulate sexual behavior in female rats, the way in which estrogen is involved remains unclear. In a long-term rat hypothalamic cell culture system, atrial natriuretic factor-producing neurons were identified as candidate sites for integration of sex steroid action. Estrogen induces the expression of progesterone receptors in atrial natriuretic factor neurons and also augments neuronal functions by increasing expression of constitutively active D5 receptors that generate cAMP in a ligand-independent manner. Such a cross-talk mechanism allows estrogen to exert its effects via the adenylyl cyclase-cAMP system by augmenting dopamine receptor activity, an action that may play an important integrative role in facilitating female sexual behavior.

From pharmacotherapy to pathophysiology: emerging mechanisms of apolipoprotein D in psychiatric disorders.

Apolipoprotein D (apoD) is an atypical plasma apolipoprotein and, based on its primary structure, it is a member of the lipocalin protein superfamily. Lipocalins have been extensively used as disease markers and, accordingly, apoD has become increasingly recognized as an important factor in the pathology of human neurodegenerative and neuropsychiatric disorders. ApoD expression is increased in the plasma and brains of subjects with schizophrenia and bipolar disorder, suggesting that it acts as a marker for disease pathology. ApoD also exhibits complex regulation by antipsychotic drug treatment and may represent a distinguishing mechanism of typical versus atypical drugs. The precise role of apoD in the CNS and disease remains to be elucidated, but recent findings have suggested that it plays an important role in the regulation of arachidonic acid signaling and metabolism providing further support for phospholipid membrane pathology in schizophrenia.

Evidence for post-translational processing of auriculin B to atriopeptin III immediately prior to secretion by hypothalamic neurons in culture.

In rats, the precursor of atrial natriuretic peptide (ANP) is produced and processed into its smaller congeners in the heart and the brain. We have demonstrated that a congener of ANP, auriculin B (ANP4-28), was present in long term monolayer cultures of neonatal rat hypothalamic neurons. In addition, forskolin and 3-isobutyl-1-methyl-xanthine (IBMX) augmented the cellular content of auriculin B in a dose dependent manner. Thus, cyclic AMP may act as an intracellular signal for modulating the functional development of hypothalamic immunoreactive (ir) ANP producing neurons. Furthermore, our data suggest that the form of ANP released from these cultures, following either forskolin treatment alone or forskolin treatment followed by acute high potassium depolarisation, was atriopeptin III (ANP5-28). Thus atriopeptin III, rather than auriculin B, may represent the endogenous ligand for ANP receptors in the central nervous system.

Glucocorticoids inhibit D1B, but not D2, receptor-mediated effects on hypothalamic atrial natriuretic factor neurons.

Recent evidence suggests that ANF neurons of the hypothalamus are dopamine sensitive, and the catecholamine may exert a direct stimulatory or inhibitory effect on the neurons mediated through D1 or D2 receptors, respectively, in a manner related to the differential dopamine binding sensitivity of the two receptor subtypes. Employing well characterized ANF RIA and colorimetric Northern blot analysis with synthetic oligonucleotide probes complementary to pro-ANF messenger RNA (mRNA), we report here the effect of dexamethasone (DM), a potent synthetic glucocorticoid, on DA-stimulated ANF neurons in long term primary cultures of neonatal rat hypothalamic cells. Although DM alone did not affect basal secretion of immunoreactive ANF, it approximately halved immunoreactive ANF secretion induced by D1 agonist, SKF38393 (P < 0.01). The effect of DM was both time dependent and dose related, with an EC50 of 0.1 nM; it was blocked by 100 nM RU38486 (P < 0.05), a glucocorticoid receptor antagonist, but not by 100 nM RU28318, a mineralocorticoid receptor antagonist. In addition, the effect of DM was mimicked by corticosterone (EC50, 10 nM), but not deoxycorticosterone. The increased expression of pro-ANF mRNA signal induced by the D1 agonist in culture was suppressed by DM in a similar manner. In contrast, DM did not modulate ANF production and secretion induced by D2 agonist, quinpirole. Furthermore, reverse transcription-polymerase chain reaction demonstrated that D1B, but not D2, receptor mRNA expression was selectively suppressed by glucocorticoids. Thus, we conclude that in monolayer cultures of rat hypothalamic neurons, glucocorticoids differentially modulate dopamine receptor-induced responsiveness of ANF neurons by down-regulating D1B, but not D2, receptor-mediated changes. Hence, in severe stress, high levels of circulating glucocorticoids may negate the D1B-induced stimulatory response but allow dopamine to suppress the function of hypothalamic ANF neurons through D2 receptor activation.

Coexpression of atrial natriuretic factor and beta-endorphin in a subpopulation of rat splenic macrophages: age-related differences.

Separate demonstration of immunoreactive (ir) atrial natriuretic factor (ANF) and ir-beta-endorphin (beta EP) in macrophages of the rat spleen prompted a reexamination of their distribution and cellular localization in addition to their developmental regulation. Double labeling immunohistochemistry was carried out on paraffin-embedded spleen sections of adult male Sprague-Dawley rats. Cells stained positive with antiserum (S118) raised against rat ANF-(99-126) were colocalized in more than 95% of cases with immunofluorescent staining of ir beta EP-(1-31) and distributed sparsely throughout the venous sinusoidal regions of the red pulp. In 1- or 5-day monolayer cultures of adherent splenocytes, 11-16% of the cells stained positive for either irANF or ir beta EP. Under these conditions, more than 95% of irANF- and ir beta EP-positive cells were also fluorescence stained for the histiocyte marker S22 or the rat macrophage marker ED-1. Colorimetric in situ hybridization similarly revealed signals for pro-ANF mRNA in more than 95% of ir beta EP-positive adherent cells. Thus, taken together with previous reports, our present findings suggest that in the rat spleen, both ANF and beta EP are produced by the same population of macrophages. However, the tissue levels and processing of the two peptides over the developmental period of the animal differed markedly in several ways. In splenic extracts of 2-day-old neonatal rats, Northern blot analysis and RIA revealed a greater abundance of pro-ANF mRNA signal and an irANF concentration about 6-fold greater than those in 30-day-old animals. In contrast, the ir beta EP concentration did not vary significantly over the same period, consistent with the level of POMC mRNA. Sephadex G-50 gel chromatography of splenic extracts from 2-day-old animals revealed predominantly higher mol wt forms of irANF and ir beta EP. From days 16-60, a significant proportion of irANF eluted in the same fractions as the mature circulating form, rat ANF-(99-126); the proportion of 3.5-kilodalton ir beta EP increased progressively to become the predominant species in adult tissues. Thus, it appears that although ANF and beta EP are coexpressed by the same population of splenic macrophages, they differ markedly during the developmental period with respect to their constitutive regulation.

Evidence for atrial natriuretic peptide-(5-28) production by macrophages of the rat spleen: an immunochemical and nonradioactive in situ hybridization approach.

Atrial natriuretic peptide (ANP), a 28-amino acid peptide, is produced and secreted by cardiac atriocytes to modulate cardiovascular functions. Recently, biologically active receptors for ANP have been demonstrated in the spleen; we report here the production of ANP-(5-28) and its 15-kDa (K) mol wt (Mr) presumptive precursors by macrophages of rat splenic tissues. Splenic, hypothalamic, and heart tissues were collected from adult male Sprague-Dawley rats and acid extracted for ANP assay. The splenic content of immunoreactive (ir) ANP (mean +/- SEM, 428 +/- 68 pg/tissue; n = 7) was approximately a fifth of that found in the hypothalamus and about 4 orders of magnitude lower than that in the heart of the same animals. The Sephadex G-50 column profile of splenic extracts revealed two immunoreactive peaks; the major peak eluted in positions consistent with 15K Mr, while a minor peak coeluted with synthetic rat ANP-(1-28) of 3K Mr. HPLC analysis of the 3K Mr species showed a single peak of immunoreactivity, which eluted with a retention time similar to that of ANP-(5-28). In rat splenic sections, immunoperoxidase localization of ir-ANP revealed positive cells sparsely distributed in marginal sinuses and the red pulp of the tissue; employing a double staining technique, S22, a surface marker for macrophages, was colocalized on the same splenocytes. Furthermore, colorimetric in situ hybridization with antisense oligonucleotide probes labeled with digoxigenin, identified specific signals for pro-ANP mRNA in splenocytes of tissue sections. In monolayer cultures of vehicle-treated splenocytes, approximately 87% of the adherent cells stained positive for S22; this marker was colocalized with ir-ANP in approximately 15% of the cells. Twenty-four-hour treatment with lipopolysaccharide (50 micrograms/ml), a bacterial endotoxin, tripled the proportion of adherent cells (32 +/- 4%; P less than 0.01) staining positive for ir-ANP over that in control cultures (mean +/- SEM, 11 +/- 3%; 10(4) cells/sample; n = 5). Furthermore, an equivalent dose of lipopolysaccharide, but not Concanavalin-A (50 micrograms/ml), quadrupled ir-ANP content compared to that in vehicle-treated cultures (less than 5 pg/well). Thus, our findings suggest that ANP-(5-28) is produced by a small population of splenic macrophages and raise the possibility that the peptide may play a signalling role at the tissue level.

Forskolin-induced immunoreactive atrial natriuretic peptide (ANP) secretion and pro-ANP messenger ribonucleic acid expression of hypothalamic neurons in culture: modulation by glucocorticoids.

In the present studies the chronic effects of glucocorticoids and drugs activating cAMP-dependent pathways on the production and secretion of immunoreactive (ir) ANP from long term monolayer cultures of neonatal rat hypothalamic neurons were examined. Forskolin treatment increased ir-ANP release in a time-dependent and dose-related manner, with an EC50 of approximately 30 microM; at a lower dose of 10 microM, forskolin doubled ir-ANP release (P less than 0.01) compared to that in control cultures (mean +/- SEM, 9.6 +/- 0.3 pg/well; n = 4). While dexamethasone (DM) alone did not affect basal secretion of ir-ANP, 10 nM of the glucocorticoid significantly enhanced the effect of forskolin (10 microM) by raising ir-ANP release approximately 3 times that induced by forskolin alone (P less than 0.001). This potentiation of DM was both time dependent and dose responsive, with an EC50 of 1 nM; this effect was significantly suppressed by 100 nM RU38486, a glucocorticoid or type II receptor antagonist, but not by RU28318, a mineralocorticoid receptor antagonist. In addition, forskolin (10 microM) or DM (10 nM) alone significantly increased ir-ANP production approximately 1.4 times (P less than 0.05) and 1.3 times (P less than 0.05) over that of control cultures, respectively, whereas concurrent treatment with forskolin and DM increased ir-ANP production by approximately 1.8 times (P less than 0.01). These changes were reflected by a corresponding increment in the abundance of pro-ANP mRNA in the cultures, as demonstrated by Northern blot analysis. We conclude from the present findings that glucocorticoid- and cAMP-dependent pathways may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.

In vitro evidence for modulation of morphological and functional development of hypothalamic immunoreactive atrial natriuretic peptide neurons by cyclic 3',5'-adenosine monophosphate.

In the hypothalamus of the rat, the precursor of atrial natriuretic peptide (ANP) is produced and processed into its smaller congeners of 3K mol wt species, which are secreted from neurons with cell bodies in the periventricular areas and the paraventricular nuclei of the tissue. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we have identified a small population of cells that stained positive for immunoreactive (ir) ANP. Seventy-two +/- 7% (mean +/- SE; n = 4 per 1000 cells) of the irANP positive cells were colocalized with the staining of neuron-specific enolase; some of the cells possessed multiple neurites and showed irANP staining in the perikarya, in the varicosities along neuronal processes, and at the terminals of long neurites. Over the range of 10(-6)-10(-4) M, forskolin, 3-isobutyl-1-methylxanthine, or 8-bromo-cAMP significantly augmented the total number of irANP-positive cells and those possessing neurites in a dose-related and time-dependent manner. At 10(-4) M, 4 days of forskolin treatment increased the number of irANP-positive neurons 4-fold (P less than 0.01) while tripling that of the cells with long neurites (P less than 0.01). Furthermore, it approximately tripled the number of cells (P less than 0.01) showing positive signals for pro-ANP mRNA, as ascertained by colorimetric in situ hybridization using a 30-basepair antisense oligonucleotide probe labeled with digoxigenin. Consistent with the above observation, forskolin, 3-isobutyl-1-methylxanthine, or 8-bromo-cAMP treatment significantly augmented the total amount of irANP present in the cultures, with an ED50 of forskolin approximating 5 x 10(-5) M. Although treatment with 10(-7) M phorbol 12-myristate 13-acetate approximately doubled the production of irANP in the cultures (P less than 0.05), phorbol 12-myristate 13-acetate had little effect on modulating the number or neurite outgrowth of irANP neurons. Thus, our present findings suggest that protein kinase-A pathways are of greater importance than protein kinase-C pathways in regulating both the functional and morphological development of ANP-producing neurons during the ontogenesis of the rat hypothalamus.

Plasticity of adrenoceptor responsiveness on irANP secretion and pro-ANP mRNA expression in hypothalamic neuron cultures: modulation by dexamethasone.

Atrial Natriuretic Peptide (ANP) or its smaller congeners are produced and secreted from the rat hypothalamus. Whereas immunoreactive (ir)ANP secretion and proANP mRNA expression in hypothalamic cell cultures of neonatal rats were augmented by norepinephrine acting through its alpha 2-adrenoceptors (AR), in the perifusion studies of adult hypothalamic fragments beta-AR was involved in the upregulation of irANP release. Here, we report that dexamethasone (DM) modulates irANP secretion and pro-ANP mRNA expression from hypothalamic neurons in culture by switching the adrenoceptor responsiveness of the cells from alpha 2- to that of beta-AR. In long term cultures of hypothalamic cells, treatment with clonidine (alpha 2-AR agonist) increased irANP secretion in a dose related manner. This effect of clonidine was abolished by DM, a glucocorticoid which by itself had little effect on the basal release of irANP. In contrast, isoprenaline, a beta-AR agonist which was ineffective when applied alone, enhanced irANP secretion from hypothalamic cultures in the presence of DM. Concurrent incubation of DM (5 nM) and isoprenaline (10 microM) augmented irANP release approximately 3 fold above that of cultures treated with DM alone (22.6 +/- 2.2; mean +/- SE, n = 4). However, phenylephrine, an alpha 1-AR agonist alone or in the presence of DM failed to stimulate irANP release. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANP mRNA in the cultures as examined by colorimetric Northern blot analysis employing a 30 mer oligonucleotide probe corresponding to the first 10 amino acid sequence of rANP1-28. We conclude from the above observations that glucocorticoids modulate irANP secretion and pro-ANP mRNA expression in hypothalamic neurons by altering the responsiveness of the cells from alpha 2-AR to that of beta-AR.

Ascorbic acid enhances forskolin-induced cyclic AMP production and pro-ANF mRNA expression of hypothalamic neurons in culture.

Besides acting as an important cofactor in the biosynthesis of catecholamine, ascorbic acid (AA) also modulates the activity of peptidyl-glycine alpha-amidating monooxygenase for the post-translational modification of neuropeptides such as alpha-MSH and TRH. We report here a novel action of AA in modulating the secretion and mRNA expression of atrial natriuretic factor (ANF) in rat hypothalamic neurons. Primary cultures of hypothalamic neurons from neonatal rats as previously described were employed in the present studies. Six days after plating, cultures were replenished with serum free media and incubated with vehicle or various doses of AA, alone or in the presence of forskolin. Treatment with AA alone significantly increased irANF secretion from the cultures in a time-related and a dose-dependent manner with an ED50 of approximately 3 microM and an Emax of 100 microM. At the concentration of 10 microM, AA augmented irANF release approximately 3 fold that of the controls (55 +/- 7 pg/well; mean +/- SE, n = 3; P < 0.01), but it failed to affect the abundance of pro-ANF mRNA in the cultures. However, 10 microM of AA markedly enhanced forskolin-induced irANF secretion and pro-ANF mRNA abundance of the cultured cells. This potentiating effect of AA on forskolin stimulation showed a good parallelism to the levels of cAMP produced in the hypothalamic cultures. We thus conclude that AA acts alone or in synergism with forskolin to stimulate the secretion and production of ANF in rat hypothalamic neurons; this latter effect may operate at the genomic level and is mediated, at least in part, through the protein kinase A dependent pathway.

Norepinephrine stimulates immunoreactive (ir) atrial natriuretic peptide (ANP) secretion and pro-ANP mRNA expression from rat hypothalamic neurons in culture: effects of alpha 2-adrenoceptors.

Although ANP or its smaller congeners are produced and secreted from rat hypothalami, the role or roles of neurotransmitter(s) in regulating their release and production from the neurons remains unclear. We report here that norepinephrine or epinephrine (NE/EPI) facilitates irANP secretion and pro-ANP mRNA expression in long term cultures of rat hypothalamic neurons through their effects on alpha 2-adrenoceptors. Hypothalami of 3 day-old Sprague-Dawley rats were removed and digested with collagenase. The dispersed cells were plated on poly-D-lysine coated culture dishes (10(6) cells/well) in Hepes buffered Dulbecco's Modified Eagle Medium supplemented with 8% fetal calf serum. Six days after plating, media were replenished with serum free media and the cultures incubated for 4 more days with vehicle or various doses of NE, EPI, alpha- or beta-adrenoceptor agonists in the presence of absence of antagonists. Culture media were then extracted with C18 Sep-pak and the levels of irANP determined by a well characterised RIA for ANP. NE or EPI treatment significantly increased irANP secretion from the cultures in a dose related manner with ED50 and Emax of approximately 0.2 microM and 1 microM respectively. The stimulation effect of NE was blocked by yohimbine (alpha 2-antagonist), but not prazosin (alpha 1-antagonist) or propranolol (beta-antagonist). Clonidine (alpha 2-agonist), but not phenylephrine (alpha 1-agonist) or isoprenaline (beta-agonist) mimicked the effects of NE or EPI. At the concentration of 0.1 microM, clonidine increased irANP release approximately 3 fold above that of control values (34.7 +/- 3.3; mean +/- SE, n = 4). These changes were accompanied by corresponding increments in the abundance of pro-ANP mRNA in the cultures as examined by a colorimetric Northern blot analysis. Our results indicate that NE or EPI, acting through its alpha 2-adrenoceptors, may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.

In vitro evidence for atrial natriuretic factor-(5-28) production by macrophages of adult rat thymi.

Although recent evidence suggests the presence of the 15-kilodalton (K) mol wt (Mr) precursor of atrial natriuretic factor (ANF) and pro-ANF mRNA in the thymus of human and rat neonates, the cellular origin of the peptides in the tissues remains to be elucidated. We report here that in adult male rats, the 15K M(r) presumptive precursor for ANF and a smaller 3K M(r), N-terminal truncated congener of the peptide, ANF-(5-28), are localized in a small population of thymic macrophages. The production of ANF in the thymus was further confirmed by demonstrating the presence of a single band of pro-ANF mRNA signal of approximately 0.8 kilobases from the tissue extract, corresponding to that in the heart. To examine the distribution of pro-ANF mRNA at a cellular level, colorimetric in situ hybridization with digoxigenin-labeled 30-mer oligonucleotides complementary to the first 10-amino acid sequence of ANF-(1-28) was employed. Positive staining was found in thymic cells localized mainly in subcapsular areas, with diffuse staining of positive cells in both the cortex and medulla mainly concentrated around the cortico-medullary junction of the tissue. The identity of the ANF-positive cells was further investigated using a double staining technique to costain immunoreactive (ir) ANF and the macrophage markers ED1 and S22 or the T-cell marker OX-19 in both thymic sections and in monolayer cultures of thymic cells. In the latter, approximately 17% of the adherent cells stained positive for irANF after 48 h in culture. Of these cells, more than 95% also stained positive for the macrophage marker ED1, whereas approximately 36% of the ED1-positive cells were colocalized with irANF. In contrast, no irANF-positive cells were colocalised with the pan-T-cell marker OX-19. In tissue sections, irANF was found in cells distributed predominantly around the cortico-medullary junctions and subcapsular areas, with diffuse staining of the cells in the medullary and cortical regions. Sephadex G-50 gel chromatographic profiles of thymic extracts revealed a major peak of immunoreactivity consistent with 15K M(r) and a smaller peak that coeluted with rat ANF-(1-28) of 3K M(r). HPLC analysis of the 3K M(r) species showed a single peak of immunoreactivity, which eluted with a retention time identical to that of synthetic rat ANF-(5-28).(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for atrial natriuretic peptide-(5-28) production by rat placental cytotrophoblasts.

Atrial natriuretic peptide (ANP), a 28-amino acid peptide, is produced and secreted by cardiac atriocytes to modulate cardiovascular and renal functions. We report here the production of ANP-(5-28) and its 15K mol wt (M(r)) presumptive precursors by cytotrophoblasts of rat placentae. Placental tissues were collected from Sprague-Dawley fetal rats on days 12, 16, 18, and 20 of gestation and acid extracted for immunoreactive (ir) ANP assay. The contents of placental irANP increased over the course of fetal growth, with the highest amount (mean +/- SE, 1083 +/- 125 pg/tissue; n = 7) found near term. Sephadex G-50 gel chromatographic profiles of the placental extract revealed a major peak of irANP coeluted with the 3K M(r) of synthetic rat (r) ANP-(1-28) and a minor peak in the position consistent with that of 15K M(r). HPLC analysis of the 3K M(r) species showed a single peak of immunoreactivity, which eluted with a retention time similar to that of rANP-(5-28). In placental sections, irANP and pro-ANP mRNA were localized by immunoperoxidase staining and colorimetric in situ hybridization in a subpopulation of placental cytotrophoblasts, but not in syncytiotrophoblasts or chorionic cells. Northern blot analysis showed a single band of pro-ANP mRNA in rat placental tissues similar in size to that found in the heart (approximately 0.85 kilobases), with the highest level of pro-ANP mRNA signal detected in the placentae of 16-day gestation fetuses. Our findings suggest that ANP is expressed and produced by a small population of rat placental cytotrophoblasts and that the 15K M(r) precursor peptide is extensively processed into the N-terminal-truncated form of ANP-(5-28) in the tissue.

A pituitary tumor producing high molecular weight adrenocorticotropin-related peptides: clinical and cell culture studies.

The tissue-specific processing of proopiomelanocortin (POMC), the precursor of ACTH, beta-endorphin, and their related peptides, is currently of considerable interest. We report a patient with a large aggressive pituitary tumor, Cushing's syndrome, and hyperpigmentation managed by transsphenoidal hypophysectomy, bilateral adrenalectomy, and sellar irradiation. Preoperatively, plasma levels of immunoreactive ACTH (ir-ACTH; 280 ng/liter) and beta-endorphin (ir-beta EP; 520 ng/liter) were moderately elevated. Chromatography of the plasma showed two peaks of ACTH immunoreactivity, with the major peak eluting in the void volume, and two major peaks of ir-beta EP, corresponding to the elution positions of beta-lipotropin and beta-endorphin standards. Plasma ir-ACTH and ir-beta EP were not suppressed by high doses of glucocorticoid or bromocriptine, a degree of autonomy more commonly found with POMC production from ectopic sources than that from pituitary tumors. Tissue removed at operation was enzymatically dispersed, and the cells were cultured in suspension, propagated, and passaged sequentially for over 20 passages. Using this cell line, we demonstrated that the biosynthesis of POMC, its pattern of processing, and the release of POMC/ir-beta EP/ir-ACTH in vitro were consistent with the in vivo evidence of autonomous secretion and abnormal processing of POMC by this pituitary tumor.

The development of a method to measure [3H] dopamine uptake by washed platelets provides no evidence for circulating inhibitors of platelet dopamine uptake in schizophrenia.

Altered [3H] dopamine uptake by platelet-rich plasma (PRP) has been reported in some subjects with schizophrenia (Rotman et al 1980; Dean et al 1990). As platelet dopamine uptake was measured using PRP, it was not possible to determine if the changes in schizophrenia were intrinsic to the platelet or due to plasma factors. Furthermore, the constraints of plasma as a medium for platelet suspension has hindered the study of the physiological requirements of platelet dopamine uptake. A method is now reported that allows platelets to be suspended in a controlled medium while preserving the dopamine uptake mechanism of the platelet. Dopamine uptake by platelets in a controlled medium was dependent on temperature, energy, sodium, and chloride. Furthermore, plasma from subjects with schizophrenia and schizophreniform disorder did not significantly alter [3H] dopamine uptake by platelets compared to the effect of plasma from control subjects. Hence, these data provide no evidence for a circulating inhibitor of platelet [3H] dopamine uptake in plasma from subjects with schizophrenia.

The binding of both [3H]nemonapride and [3H]raclopride is increased in schizophrenia.

We performed a postmortem autoradiographic study to compare the density of dopamine D4-like sites in caudate putamen section from age-matched schizophrenia (n = 15) and control (n = 15) populations. The densities of the D4-like sites were estimated by subtracting the density of [3H]raclopride (binding D2 and D3 receptors) from the density of [3H]nemonapride (binding D2, D3, and D4 receptors). The findings revealed that in the schizophrenia population there was a significant increase in the binding of both [3H]nemonapride (2.3-fold) and [3H]raclopride (1.9-fold). In addition, in the schizophrenia population the density of D4-like sites was increased 2.6-fold.