Hypothalamic D2 receptors mediate the preferential release of somatostatin-28 in response to dopaminergic stimulation.

We have studied the effect of dopamine (DA) together with agonist and antagonist drugs of varying specificity on the release of immunoreactive forms of somatostatin (SS) from the perfused, adult rat hypothalamus in vitro. Levels of SS increased from 14.7 +/- 3.7 pg (mean +/- SE) under basal conditions to 137 +/- 23.0 pg after exposure to 10(-6) M DA. This dopaminergic effect was mimicked by the specific D2 agonists bromocriptine (10(-7) M) and LY 171555 (10(-6) M) but not by the D1 agonist SKF 38393A (10(-6) M). The stimulatory action of DA (10(-6) M) was blocked by the active (d) but not the inactive (l) isomer of butaclamol (10(-7) M). Similar blockade was achieved with the specific D2 antagonists metoclopramide (10(-8) M) and domperidone (10(-8) M), whereas the D1 antagonist SCH 23390 partially blocked the stimulation of DA but only when used at X100 greater concentration (10(-6) M). SCH 23390 (10(-8) M) did not affect the dopaminergic stimulation of SS release. HPLC characterization of the immunoreactive forms of SS yielded two peaks which corresponded to SS-28 and SS-14. The ratio of these forms varied significantly under different conditions. In the basal state the ratio of SS-28 to SS-14 was 1:4.4; in response to stimulation with DA, the ratio was 1:1.7 and in response to depolarization with 60 mM K+ the ratio was 1:3.1. In conclusion, the stimulatory action of DA on SS release is mediated via hypothalamic D2 receptors. Furthermore dopaminergic stimulation increases the molar ratio of SS-28 to SS-14 in the total immunoreactive SS which is released.

Adenosine-regulated cell proliferation in pituitary folliculostellate and endocrine cells: differential roles for the A(1) and A(2B) adenosine receptors.

A(1) and A(2) adenosine receptors have been identified in the pituitary gland, but the cell type(s) on which they are located and their effects on pituitary cell growth are not known. Therefore, we analyzed the expression of A(1) and A(2) receptors in primary rat anterior pituitary cells, two pituitary folliculostellate (TtT/GF and Tpit/F1) and two pituitary endocrine (GH(3) and AtT20) cell lines, and compared their effects on cell proliferation. In anterior pituitary and folliculostellate cells, adenosine and adenosine receptor agonists (5'-N-ethylcarboxamidoadenosine, a universal agonist, and CGS 21680, an A(2A) receptor agonist) stimulated cAMP levels with a rank order of potency that indicates the presence of functional A(2B) receptors. This stimulation, however, was not observed in either GH(3) or AtT20 cells, where adenosine and the A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine inhibited VIP/forskolin-stimulated cAMP production. Expression of A(2B) and A(1) receptors in the folliculostellate cells and that of the A(1) receptor in the endocrine cells were confirmed by RT-PCR, immunocytochemistry, and ligand binding. Adenosine and 5'-N-ethylcarboxamidoadenosine dose-dependently (10 nM to 10 microM) stimulated growth in the folliculostellate, but not in the endocrine, cells, whereas in the latter, 100 microM adenosine and 2-chloro-N(6)-cyclopentyladenosine inhibited cell proliferation by slowing cell cycle progression. These data highlight the differential expression of A(1) and A(2B) adenosine receptors in pituitary cells and provide evidence for opposing effects of adenosine on pituitary folliculostellate and endocrine cell growth.

Immortalized human pituitary cells express glycoprotein alpha-subunit and thyrotropin beta (TSH beta).

A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.

Piperidinyltetralin sigma ligands.

Sigma receptor ligands have been proposed to be potential antipsychotic drugs based on their activity profile in animal behavioral models and their indirect modulation of dopaminergic function. Compound 15 (DuP 734) is a combined antagonist of sigma-1 and serotonin 5HT2 receptors, which has been entered into phase I clinical trials as a potential antipsychotic drug. Tetralins 1 and 2 were prepared to determine whether restriction of the conformation of 15 and its analogs may lead to differences in binding selectivity or in vivo profile. The syntheses and the structure-activity relationships of these compounds are reported herein. A reduced derivative, 14, had high affinity for sigma-1 and serotonin 5HT2 receptors as well as excellent oral activity in some animal antipsychotic models. Furthermore, compound 14 failed to cause catalepsy in the rat up to 90 mg/kg (po).

Dopamine stimulates release of thyrotrophin-releasing hormone from perfused intact rat hypothalamus via hypothalamic D2-receptors.

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 mumol/l (increase over basal, 118 +/- 16.5 (S.E.M.) fmol TRH; P less than 0.001 vs basal). This effect was mimicked by the specific D2-agonist drugs bromocriptine (0.1 mumol/l) and LY 171555 (0.1 mumol/l) (increase over basal values, 137.5 +/- 13.75 fmol and 158.6 +/- 10.7 fmol respectively; P less than 0.001 vs basal), but not by the D1-agonist SKF 38393A. The stimulatory effect of dopamine (1 mumol/l) was blocked in a stereospecific manner by the active (D) but not by the inactive (L) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D2-antagonist domperidone (0.01 mumol/l) whereas the D1-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0.01 mumol/l) of this D1-antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D2-receptors.

Effects of thyroid status on brain catecholamine biosynthesis in adult rats: assessment by a steady-state method.

Effects of thyroid status on brain catecholamine turnover in adult rats were investigated using a steady-state method. Rats were treated for 3 weeks with s.c. injections of L-thyroxine (0.4 mg/kg), aminotriazole in drinking water (0.1%, w/v) or vehicle. After 2 weeks of treatment rats were implanted chronically with lateral intracerebroventricular (i.c.v.) cannulae. They were injected i.c.v. with [3H]tyrosine 1 week later. Catecholamine and tyrosine content and specific activity were measured in mediobasal hypothalamus, anterior hypothalamus and striatum, using high-performance liquid chromatography with electrochemical detection. Thyroxine treatment resulted in a significant increase in noradrenaline and dopamine synthesis localized to the mediobasal hypothalamus. Conversely, aminotriazole treatment resulted in a significant decrease in noradrenaline synthesis localized to the mediobasal hypothalamus. The localization of these changes in catecholamine turnover to the mediobasal hypothalamus suggests that they may be specific functional effects which are of importance in the overall integrated control of thyroid function.

Desensitisation of somatostatin, TRH and GHRH responses to glucose in the diabetic (Goto-Kakizaki) rat hypothalamus.

We have studied the effects of glucose on the release of somatostatin (SS), TRH and GHRH from incubated hypothalami of normal and genetically diabetic, Goto-Kakizaki (GK) rats. The active isomer D-glucose caused a dose-related inhibition of SS, TRH and GHRH from normal rat hypothalami over a 20-min incubation period in vitro. In contrast, in GK rats the effects of glucose on TRH and SS were significantly reduced and the effects on GHRH were abolished. These data indicate that the sensitivity of SS-, TRH- and GHRH-producing hypothalamic neurones is reduced in diabetic rats. The effect is most pronounced for GHRH release as there was no change in the release of this peptide with increasing glucose concentrations. In conclusion, it appears that the diabetic state in GK rats causes differential desensitisation (GHRH > TRH and SS) of neuronal responses to subsequent changes in glucose concentrations in vitro. This may be due to alterations in the neurotransmitter control and/or a reduction in number, affinity or function of glucose transporters on these peptidergic neurones or other intermediary neuronal pathways.

Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence.

The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5' end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5' truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5' truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5' truncated POMC gene occurring in pituitary cells.

Development and validation of a two-site immunochemiluminometric assay for rat growth hormone-releasing hormone.

Abstract We describe the development and validation of a two-site immunochemiluminometric assay for rat growth hormone-releasing hormone (GHRH) based on the affinity purification of polyclonal rabbit antisera to rat GHRH using a human 1-29 GHRH affinity column. Assay sensitivity is 3.2 pg/ml using 100 mul of unextracted sample and the working range for the assay within 15% confidence limits is 64 to 5,000 pg/ml. Rat hypothalamic extract and secreted material demonstrated a single large peak of immunoreactive material coeluting with synthetic rat GHRH on high-performance liquid chromatography with a smaller, earlier peak which probably represents methionine sulphoxide [Met(O)(27)] GHRH. Extracted material diluted in parallel to the standard curve. Incubated rat hypothalami readily released measurable amounts of rat GHRH which responded appropriately to depolarization with 60 mM K(+) in a Ca(2+)-dependent manner.

Glutamate pathways mediate somatostatin responses to glucose in normal and diabetic rat hypothalamus.

We investigated the role of hypothalamic glutamate receptors in mediating the stimulatory effect of low glucose (< 5 mM) on somatostatin release. We also studied whether alteration in glutamate release might contribute to the reduced hypothalamic somatostatin response to low glucose observed in diabetic (Goto-Kakizaki) rat hypothalami. Hypothalamic somatostatin release in response to incubation with 1 mM D-glucose was inhibited by the ionotropic glutamate receptor antagonists MK801, D-AP5 and DNQX but not by the metabotropic antagonists L-AP3 or MCPG. The release of somatostatin was increased by the ionotropic agonists NMDA, AMPA and kainate but not by metabotropic agonists t-ACPD or L-AP4. Basal and peak glutamate release in response to incubation with 1 mM glucose, were significantly lower from GK hypothalami There were no significant differences in the basal or stimulated release of serine and GABA. These data indicate that ionotropic NMDA/AMPA/kainate receptors and not metabotropic receptors mediate the effects of glucose on rat hypothalamic somatostatin release. Reduced hypothalamic somatostatin release in response to low glucose in diabetic (Goto-Kakizaki) rats may well be secondary, at least in part, to reduced glutamate release.

Mitogen-activated protein kinase mediates epidermal growth factor-induced morphogenesis in pituitary GH3 cells.

Epidermal growth factor (EGF) causes pituitary GH3 cells to change from their normal predominantly rounded morphology to much more elongated cells with extensive filopodia, and this effect is accompanied by a parallel increase in cell volume. In view of this, and because EGF receptor expression is increased in some pituitary tumours, we examined the mechanism of this EGF-induced morphological effect as it may play a role in tumour invasiveness. The effect of treatment of the cells with EGF (1 nm, 4 days) was determined visually (expressed as percent non round cells) and by measuring the cell volume by Coulter Counter analysis. EGF treatment caused the cells to change their morphology with percent non round cells increasing from 37% in control cells to 74% in EGF-treated cultures; this was accompanied by a parallel increase in cell volume. Treatment of the cells with EGF in the presence of the MEK1 inhibitor (PD98059) completely blocked the EGF-induced morphological changes, showing that activation of the mitogen-activated protein kinase (MAPK) pathway is necessary to mediate this effect. Transfection of the cells with a constitutively activated mutant of MEK1 produced a similar morphological change to that produced by EGF treatment, with the proportion of non round cells increasing to 62% with a parallel increase in cell volume compared to cells transfected with the empty vector, demonstrating that direct activation of MAPK pathway is sufficient to mediate the observed morphological effects. The effects produced by activated MEK1 transfection could be blocked by PD98059. EGF had opposing effects on prolactin and growth hormone (GH) secretion by the cells, increasing prolactin release and inhibiting GH release. Transfection of the cells with activated MEK1 produced similar effects on hormone release as EGF treatment. In conclusion, the morphological effects of EGF on GH3 cells are mediated by activation of the MAPK pathway as blockade of this pathway abolished the observed effect, and direct activation of this pathway by transfection with an activated mutant of MEK1 was able to duplicate these effects. This mechanism may contribute to the growth and possibly local invasiveness of some pituitary tumours that express the EGF receptor.

Effects of Glucose on Thyrotrophin-Releasing Hormone, Growth Hormone-Releasing Hormone, Somatostatin and Luteinizing Hormone-Releasing Hormone Release from Rat Hypothalamus in vitro.

Abstract Increasing concentrations of D-glucose (1 to 25 mM) inhibited somatostatin, thyrotrophin-releasing hormone (TRH) and growth hormone-releasing hormone (GHRH) release from incubated adult rat hypothalami in a stereospecific manner. In contrast, the effects of D- and L-glucose on luteinizing hormone-releasing hormone release were virtually identical. Increasing concentrations of D-glucose also inhibited somatostatin release following depolarization with high K(+), but had no obvious effect on depolarization-induced TRH or GHRH release when compared with L-glucose. In conclusion, D-glucose exerts a potent, dose-related modulatory action on the release of rat hypothalamic TRH and GHRH as well as somatostatin in vitro. Further studies are required to establish any physiological relevance of glucose in the modulation of these hypothalamic neuropeptides.

An in vivo steady-state method for the determination of catecholamine biosynthesis in the rat brain using high-performance liquid chromatography with electrochemical detection.

A technique is described for the measurement of steady-state catecholamine (CA) synthesis in the rat brain in vivo, using [3H]tyrosine incorporation with high-performance liquid chromatography (HPLC) and electrochemical detection. Adult male rats chronically implanted with lateral intracerebroventricular (i.c.v.) cannulas, were injected i.c.v. with [3H]tyrosine. CA and tyrosine content and specific activity were measured in mediobasal hypothalamus, anterior hypothalamus and striatum. A time-dependent increase in CA synthesis occurred in all tissues over 20 min post-i.c.v. injection. The technique described may prove to be useful in the assessment of central neurotransmitter turnover in various physiological and pharmacological settings.

The treatment of hypertension with verapamil.

In a randomised double blind cross over trial, verapamil produced significant dose dependent reductions in blood pressure in 23 patients. There were minimal side-effects. All patients had some contraindication to the use of beta blocker therapy, and the role of verapmil in this situation is discussed.

Pitfalls of spirometry.

Many spirograms cannot be interpreted properly even when the technician is expert. The criteria of an interpretable spirogram are (1) full inspiration, (2) quick attainment of highest flow, (3) continuous decrease in flow with expiration, (4) smooth, gradual termination, and (5) expiration lasting three seconds or more. Violation of these criteria leads to "pseudo-obstruction" when expiration is not forceful, to "concealed obstruction" when the graphic record is started late and to two kinds of "pseudo-restriction," one due to inadequate inspiration and the other to premature termination of expiration. A "decision tree" for dealing with large volumes of spirometric data is presented.

Workers with multiple chemical sensitivities: psychosocial intervention.

Treatment of MCS, an illness characterized by reaction to a multiplicity of factors coming from within the patient and from the social and physical environment, must incorporate multiple types of help, all directed toward supplying what these patients require. Medical, psychiatric, and social work treatment are all significant and all different, with overlap in several areas. As in all practice in the medical setting, the overall function of the social worker is to enable the MCS patient to make use of what the physician has to offer by supporting the patient's capacity to cope with the social and emotional impact of his/her illness.

Measurement of arterial blood gases at the transition from exercise to rest.

Arterial blood gas samples obtained 5-20 s after stair-climbing exercise were compared with samples taken during the last 30 s of exercise in 137 subjects. Arterial partial pressure of CO2 (PaCO2) did not change significantly, and in 110 subjects the two samples were within the analytical variation (+/- 2 Torr), supporting the cardiodynamic hypothesis of respiratory regulation. Exceptions to this response were 10 subjects who hyperventilated (PaCO2 less than 34) during exercise and 15 with severe obstruction [forced expiratory volume in 1 s (FEV1) less than 70% forced vital capacity (FVC), and FVC less than 70% of predicted] in whom PaCO2 increased significantly. Overall, arterial partial pressure of O2 (PaO2) increased an average of 3.49 Torr (P less than 0.001). In the two groups in which PaCO2 increased, postexercise PaO2 did not rise. In addition, duration of exercise affected PaO2 response. PaO2 increased significantly more after brief (less than 2 min) periods than after longer (4-6 min) exercise, and this difference increased only when subjects with normal or borderline ventilatory function were analyzed. In 13 subjects in whom a second sample was taken 30-45 s after exercise, the increase in PaO2 was progressive and again the difference between short and long exercise was evident. Regulation of respiration to maintain PaCO2 and changes in O2-CO2 kinetics, leading to an increase in the gas exchange ratio at the exercise-rest transition, are the most likely explanations of these data which establish that the usual response to stopping exercise in normal subjects and most patients is an unchanged PaCO2 and a variable increase in PaO2.

Adipose tissue cellularity: effect on insulin and thyroxine.

The influence of insulin and thyroxine on the cellularity of adipose tissue in the rat epididymal fat pad has been studied. Incorporation of (3H) thymidine into the DNA of fat cells and stroma was measured together with fat cell size and number in rats pre-treated with either one of these hormones. There was an increase in fat pad weight in insulin treated rats which was due to 'lipid filling' of existing adipocytes and not increased proliferation of new fat cells. Thyroxine treated rats showed a decrease in fat pad weight caused by a decrease in size of individual fat cells. Cell number remained unaffected by either treatment.

Routine pulmonary function tests during bleomycin therapy. Tests may be ineffective and potentially misleading.

Forced vital capacity (FVC) and diffusing capacity for carbon monoxide (DLco) studies in two patients with proved interstitial fibrosis after low doses of bleomycin were contrasted with these measurements in 21 patients who received bleomycin without toxicity. The FVC decreased significantly (20% or more) in both fibrosis patients and in seven others. In one fibrosis patient and in three others the results could be explained by weakness. The DLco fell in both patients with fibrosis and in 12 others. Correction for decrease in hemoglobin level accounted for the change in one of the toxic patients and seven of the others, but hemoglobin correction made two insignificant changes significant and increased therapy values above control four times. Since these tests have many false-positive results and can be affected by weakness and anemia leading to false-negative results their use is ineffective and may be misleading.