Andrew Levy

p53 gene mutations in pituitary adenomas: rare events

OBJECTIVE Occult pituitary adenomas are said to occur in up to 20% of random autopsy examinations, yet the only oncogene known to be associated with pituitary adenomas, gsp, is found in only 40% of somatotrophinomas, a subtype that accounts for a minority of pituitary tumours. Mutations of the p53 tumour suppressor gene are thought to be involved in the pathogenesis of as many as 50% of all human cancers, including tumours of the central nervous system. The objective of this study was to determine whether p53 gene mutations are associated with pituitary adenomas.

Anterior pituitary cell population control: basal cell turnover and the effects of adrenalectomy and dexamethasone treatment

We have used an extremely accurate, dedicated, real time computerized image analysis system to facilitate the manual quantification of changes in the prevalence of mitotic figures and apoptotic bodies in male rat pituitary following surgical adrenalectomy and, 14 days later, dexamethasone treatment. Under basal conditions, the prevalence of mitotic figures and apoptotic bodies was 0.066±0.016% and 0.030±0.012% (mean±SE) respectively. Dexamethasone treatment reduced the prevalence of mitotic figures and, in adrenalectomized animals, produced a highly significant and reproducible burst of apoptotic activity that peaked 48u2003h after the beginning of treatment (0.261±0.022%) before falling sharply to control levels within a further 8u2003h. Two weeks after the start of dexamethasone treatment, total pituitary cell numbers continued to decline. The rate of accumulation of mitotic figures in vivo after colchicine treatment indicates that mitosis is histologically overt in 2u2003μm thick hematoxylin and eosin stained sections under the light microscope for around 80u2003min; that apoptosisu2003—u2003identified as classical apoptotic bodiesu2003—u2003is overt for 44u2003min and that, on average, a young, adult, male rat anterior pituitary cell either dies or divides as frequently as once every 60–70 days. These data show that transient and apparently trivial fluctuations in the prevalence of apoptotic and mitotic events have a profound effect on pituitary cell population dynamics, and demonstrate that dexamethasone treatment of adrenalectomized rats produces a decline in total anterior pituitary cell numbers that continues for at least 2 weeks after the start of glucocorticoid treatment.

WATER DEPRIVATION IN THE RAT INDUCES NITRIC OXIDE SYNTHASE (NOS) GENE EXPRESSION IN THE HYPOTHALAMIC PARAVENTRICULAR AND SUPRAOPTIC NUCLEI

We examined the effects of water deprivation on nitric oxide synthase (NOS) gene expression in the rat hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. Dehydration caused a significant increase in NOS gene transcripts in the PVN and SON but not in the subfornical organ (SFO). The results suggest that dehydration has a major effect on the NOS gene expression in the PVN and SON.

NUCLEOTIDES AS EXTRACELLULAR SIGNALLING MOLECULES

There is now wide acceptance that ATP and other nucleotides are ubiquitous extracellular chemical messengers. ATP and diadenosine polyphosphates can be released from synaptosomes. They act on a large and diverse family of P2 purinoceptors, four of which have been cloned. This receptor family can be divided into two distinct classes: ligand‐gated ion channels for P2X receptors and G protein‐coupled receptors for P2Y, P2U, P2T and P2D receptors. The P2Y, P2U and P2D receptors have a fairly wide tissue distribution, while the P2X receptor is mainly found in neurons and muscles and the P2T and P2Z receptors confined to platelets and immune cells, respectively. lnositol phosphate and calcium signalling appear to be the predominant mechanisms for transducing the G‐protein linked P2 receptor signals. Multiple P2 receptors are expressed by neurons and glia in the CNS and also in neuroendocrine cells. ATP and other nucleotides may therefore have important roles not only as a neurotransmitter but also as a neuroendocrine regulatory messenger.

Rapid changes of heteronuclear RNA for arginine vasopressin but not for corticotropin releasing hormone in response to acute corticosterone administration.

Corticotropin‐releasing hormone and arginine vasopressin gene transcription were studied in the parvocellular cells of the hypothalamic paraventricular nucleus in response to both adrenalectomy and the injection of corticosterone. Following 6 days ADX, there was an increase in AVP mRNA, AVP hnRNA and CRH mRNA but not CRH hnRNA. After injection of corticosterone in ADX rats there was an extremely rapid reduction in AVP hnRNA which fell markedly within 15min. AVP mRNA, however, remained raised throughout the 4 h. CRH hnRNA fell at 2 and 4u2003h, while CRH mRNA was only significantly reduced at the 4u2003h time point. Data demonstrate markedly different kinetics for the glucocorticoid regulation of the AVP and CRH genes in the hypothalamus.

Hypovolemia upregulates the expression of neuronal nitric oxide synthase gene in the paraventricular and supraoptic nuclei of rats

We have examined the effects of isotonic hypovolemia on the expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) of the rat, using in situ hybridization histochemistry with a 35S-labelled oligodeoxynucleotide probe complementary to nNOS mRNA. Intraperitoneal (i.p.) administration of polyethylene glycol (PEG) (MW 4000, 20 ml/kg body weight) dissolved in 0.9% saline (20% w/v) induced isotonic hypovolemia. The expression of the nNOS gene in the PVN and SON 6 h after i.p. administration of PEG was increased significantly in comparison with controls. The dual staining for NADPH diaphorase activity and Fos-like immunoreactivity (Fos-LI) showed that at 3 and 6 h after i.p. administration of PEG, a subpopulation of NADPH diaphorase-positive cells in the PVN and SON exhibited nuclear Fos-LI. These results suggest that NO in the PVN and SON may be involved in the neuroendocrine and autonomic responses to non-osmotic hypovolemia.

Cloning and expression of melanin-concentrating hormone genes in the rainbow trout brain

Salmonids, a group of tetraploid fish including salmon and trout, produce the vertebrate neuropeptide melanin-concentrating hormone (MCH) in a group of hypothalamic magnocellular neurons in the nucleus lateralis tuberis (NLT). NLT neurons project both to the brain and to the neural lobe of the pituitary gland from where MCH is released into the circulation to play a central role in camouflage (+/- stress). We have cloned and sequenced the MCH1 and MCH2 genes from the rainbow trout, Oncorhynchus mykiss, and used the data firstly to examine the position of O. mykiss in salmonid phylogeny, and secondly to enable central nervous system MCH1 and MCH2 gene expression to be mapped. In the immature adult female trout brain, only MCH2 was detectable at the hybridization stringency used. In addition to the known location of MCH-positive neurons, immunocytochemistry and in situ hybridization histochemistry revealed a previously undescribed nucleus of MCH-positive neurons located more dorsal and posterior to those of the NLT, over the paraventricular organ of the lateral ventricular recess. Axons from this second group of MCH neurons project dorsally into the brain, while a few extend down toward the lateral ventricle near the paraventricular organ. They make little, if any, direct contact with the neurohypophysis, and thus may subserve a central function, unrelated to hormonal colour regulation.

Inhibition of the Hypothalamic-Pituitary-Thyroid Axis in Response to Lipopolysaccharide Is Independent of Changes in Circulating Corticosteroids

We have investigated the role of circulating glucocorticoids in the suppression of the hypothalamic-pituitary-thyroid (HPT) axis following lipopolysaccharide (LPS) injection in rats. Intraperitoneal injection of LPS (2.5 mg/kg) suppressed paraventricular nucleus thyrotropin-releasing hormone (TRH) mRNA, pituitary thyroid-stimulating hormone (TSH) mRNA and plasma triiodothyronine. In these animals LPS also increased paraventricular nucleus corticotropin-releasing hormone (CRH) mRNA, pituitary proopiomelanocortin (POMC) mRNA and plasma corticosterone levels. To investigate the role of plasma corticosterone in the suppression of the HPT axis, we clamped the plasma corticosterone level at morning baseline level by bilateral adrenalectomy and corticosterone pellet implantation. Ten days after surgery, LPS injection evoked a dramatic increase in CRH mRNA and POMC mRNA. Despite the lack of change in plasma corticosterone in the corticosterone-clamped rats, LPS was still able to suppress TRH and TSH mRNA levels in both corticosterone-clamped and sham-operated rats. These data indicate that in response to LPS, suppression of the HPT axis occurs and is independent of the LPS-induced increase in plasma corticosterone.

Influence of Environmental Colour and Diurnal Phase on MCH Gene Expression in the Trout

Melanin‐concentrating hormone (MCH) gene expression in the brain of rainbow trout, reared and maintained in either pale or black‐coloured tanks, was studied using in situ hybridization histochemistry. MCH transcripts were most prevalent in the magnocellular neurones of the nucleus lateralis tuberis (NLT), which project to the pituitary gland. They were also present, although at much lower levels, in dorsally projecting parvocellular neurones, sited more posteriorly above the lateral ventricular recess (LVR). In the NLT the most intense hybridization signal was seen over the pituitary stalk; above the LVR, the most active neurones were located caudally. In both the NLT and above the LVR, MCH hybridization signal was 4‐fold stronger in white‐reared fish than in black‐reared fish. There was also a marked diurnal variation in MCH expression in both sites, with high levels at 16.00 h and lower levels at 04.00 h. The results show that gene activity in both hormonal (NLT) and neuromodulator/neurotransmitter (LVR) MCH neurones is induced by pale environmental colour and that MCH gene activity is subject to pronounced diurnal variation.

Inhibition of Hypothalamic Nitric Oxide Synthase Gene Expression in the Rat Paraventricular Nucleus by Food Deprivation is Independent of Serotonin Depletion

We have investigated the effects of food deprivation on nitric oxide synthase (NOS) transcript levels in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. Food deprivation for 48 h significantly and consistently reduced NOS transcript prevalence by approximately 50% in both sites. Since there is considerable evidence for an important role of 5‐HT in feeding behaviour, we then examined the effect of food deprivation on NOS gene expression in the PVN following para‐chlorophenylalanine (PCPA)‐induced hypothalamic 5‐HT depletion. As starvation causes central down‐regulation of the thyroid axis, changes in thyrotropinreleasing hormone (TRH) and pituitary thyrotrophin (TSH) transcript prevalence were used as internal controls. PCPA pretreatment (200 mg/kg body weight as a single daily dose ip for 2 days) had no significant effect on basal levels of NOS, TRH or TSH transcripts, or on the effect of a subsequent 48 h fast, which significantly reduced all three. These results show for the first time, that food deprivation for 48 h significantly reduces NOS gene expression in the rat PVN and SON. Secondly, that basal levels and the fasting‐induced reductions in the prevalence of NOS, TRH and TSH transcripts were not affected by PCPA‐induced hypothalamic 5‐HT depletion. Therefore, at least under the experimental conditions used here, 5‐HT does not appear to be involved in setting baseline levels—or in the starvation‐induced inhibition of NOS or thyroid axis gene expression in the PVN.