Hardial S. Chowdrey

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.

Chronic Administration of Glucocorticoids Directly Upregulates Prepro‐Neuropeptide Y and Y1‐Receptor mRNA Levels in the Arcuate Nucleus of the Rat

The complete sequence of the cDNA encoding the neuropeptide Y (NPY) Y1‐receptor has recently been deduced from a rat brain library, and the presence of messenger ribonucleic acid (mRNA) encoding Y1‐receptor protein has been demonstrated within the brain. Using quantitative in situ hybridization histochemistry, the content and distribution of Y1receptor and preproNPY mRNAs have been investigated in the hypothalamic arcuate nucleus of adrenalectomized rats receiving glucocorticoid replacement therapy for 12 days by means of either high doses of dexamethasone in their drinking water or by subcutaneous corticosterone pellets. Basal metabolic parameters such as weight gain or loss, blood glucose and plasma insulin were monitored: Dexamethasone treatment induced weight loss and a state of hyperinsulinemia with normoglycemia, while corticosterone treated animals displayed metabolic parameters identical to sham ADX animals. Within the arcuate nucleus of glucocorticoid treated animals, levels of Y1receptor and preproNPY mRNAs were increased. In contrast, adrenalectomy itself had no effect upon Y1‐receptor mRNA levels or preproNPY mRNA levels in the arcuate nucleus. These studies demonstrate that glucocorticoids exert a stimulatory action on levels of Y1‐receptor mRNA and preproNPY mRNA levels in the hypothalamic arcuate nucleus. This is the first evidence to suggest that the expression of a neuropeptide‐receptor gene in the central nervous system may be directly sensitive to peripheral hormonal signals.

The effects of restraint or hypertonic saline stress on corticotrophin-releasing factor, arginine vasopressin, and proenkephalin A mRNAs in the CFY, Sprague-Dawley and Wistar strains of rat.

It is generally assumed that the stress response of different strains of rat will be identical following exposure to acute stress. In the present study we have examined the activation of the hypothalamo-pituitary-adrenal axis in the Wistar, Sprague-Dawley and CFY strains of rat following exposure to either the predominantly psychological stress of restraint or the physical stress of i.p. hypertonic saline injection. We have investigated the hypothalamic activation of corticotrophin-releasing factor (CRF) and proenkephalin A (PEA) mRNAs in the parvocellular cells of the paraventricular nucleus (PVN) and arginine vasopressin (AVP) in both the magnocellular and parvocellular regions in the PVN following acute stress. In addition we have measured corticosterone as an index of end-point activation. Circulating corticosterone and CRF mRNA were increased in all three strains following either stress. AVP and PEA mRNAs were increased following hypertonic saline but only in the CFY strain following restraint. Overall the relative increase in the parameters measured was greater in the CFY strain of rat than the other strains. These data demonstrate marked differences in response to acute stress in the three strains of rat examined. These varying responses must be taken into consideration when designing or interpreting any study investigating the stress response.

Contents of corticotropin-releasing hormone and arginine vasopressin immunoreactivity in the spleen and thymus during a chronic inflammatory stress.

We have previously found that proopiomelanocortin (POMC) mRNA and levels of adrenocorticotropin (ACTH) and beta-endorphin peptides are increased in the spleen and thymus of rats with adjuvant-induced arthritis (AA), and immunologically mediated inflammatory disease. To determine whether alterations in immune tissue POMC during AA are also accompanied by changes in immune tissue corticotropin-releasing hormone immunoreactivity (ir-CHR) and arginine vasopressin (AVP), we measured ir-CRH and AVP by radioimmunoassays in spleen and thymic extracts 14 days following injection of adjuvant. Ir-CRH was detectable in all extracts of spleen and thymus. Total contents of ir-CRH in the spleen and thymus were not altered following arthritis, although a significant decrease was observed in splenic extracts from arthritis rats (40.0 +/- 4.2 fmol/g tissue) compared to controls (69.5 +/- 8.4 fmol/g tissue) when contents were expressed as amount per weight of tissue. Low levels of AVP were also detected in immune tissues, with contents significantly increased in spleens from arthritis animals (17.4 +/- 1.6 fmol/g tissue) compared to controls (10.6 +/- 1.9 fmol/g) but thymic contents of AVP were not altered by arthritis (10.6 +/- 1.3 fmol/g) compared to controls (9.2 +/- 0.7 fmol/g). Control levels of AVP were significantly higher in spleens and thymuses from female rats (53 +/- 5 and 25 +/- 4 fmol/g tissue, respectively) compared to males. G-50 chromatography revealed that the principal form of splenic ir-CRH is CRH(1-41), although in non-arthritic animals some ir-CHR eluted in a position indicating a slightly larger form.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The diurnal expression of genes encoding vasopressin and vasoactive intestinal peptide within the rat suprachiasmatic nucleus is influenced by circulating glucocorticoids

The mammalian suprachiasmatic nucleus (SCN) is the endogenous pacemaker generating the diurnal rhythm of the stress hormones ACTH and glucocorticoid secretion. In the present study, we have employed male rats entrained to a 12:12 h (light:dark) photoperiod to investigate the effects of chronic and acute administration of exogenous glucocorticoids upon the diurnal expression of vasopressin and vasoactive intestinal peptide (VIP) mRNA in the SCN by semiquantitative in situ hybridization histochemistry. Chronic administration of exogenous glucocorticoids significantly enhanced vasopressin mRNA expression only at zeitgeber time (ZT) 5, while the otherwise rhythmic expression of vasopressin mRNA was unaffected at ZT11, ZT17 and ZT23. In contrast, the same treatment abolished the rhythmic expression of VIP mRNA resulting in constantly elevated mRNA levels. In adrenalectomized rats given an overnight supplement of dexamethasone in their drinking water, the expression of both vasopressin and VIP mRNA in the SCN was elevated the following morning at ZT6 when compared to adrenalectomised rats kept on 0.9% saline. These results suggest that glucocorticoids influence the expression of vasopressin during a narrow window of time in the diurnal cycle coinciding with the time where entrainment of the circadian pacemaker with non-photic cues is possible. Constantly elevated levels of glucocorticoids may also interfere with the suprachiasmatic expression of VIP mRNA which is thought to be driven by photic cues.

Effects of a chronic inflammatory stress on levels of pro-opiomelanocortin-derived peptides in the rat spleen and thymus

Adjuvant-induced arthritis (AA) in specific strains of rats is an immunologically mediated inflammatory disease which is also characterised by activation of the endocrine system. To further investigate the effects of AA on processing of the pro-opiomelanocortin (POMC) precursor in rat immune tissues, we utilised radioimmunoassays for adrenocorticotrophin (ACTH), beta-endorphin and alpha-melanocyte-stimulating hormone (alpha-MSH) to measure these peptides in the spleen and thymus. 14 days following adjuvant injection, spleen levels of ACTH were elevated in the AA group (4.47 +/- 1.04 ng/g tissue, n = 9) compared to controls (2.42 +/- 0.4 ng/g) and exacerbation of the disease by removal of circulating glucocorticoids through bilateral adrenalectomy (ADX) resulted in further elevation of spleen ACTH (5.11 +/- 1.22 ng/g). beta-Endorphin levels in both the AA (10.60 +/- 1.61 ng/g) and AA/ADX (13.37 +/- 2.36 ng/g) groups were higher than controls (5.57 +/- 0.65 ng/g). Conversely, alpha-MSH spleen levels were decreased in the AA (2.89 +/- 0.22 ng/g) and AA/ADX (2.22 +/- 0.33 ng/g) groups compared to controls (4.62 +/- 0.45 ng/g) and were also decreased following adrenalectomy. In the thymus, ACTH levels were elevated in the AA group (8.95 +/- 1.41 ng/g) compared to controls (5.79 +/- 0.63 ng/g), and the same pattern was evident for thymic alpha-MSH (0.64 +/- 0.08 ng/g in AA animals compared to control levels of 0.35 +/- 0.03 ng/g). Following G50 gel filtration, ACTH and beta-endorphin immunoreactivities (ir) were present in both spleen and thymus as two peaks, one which eluted near the void volume and one which eluted in a lower molecular mass position than the standards.(ABSTRACT TRUNCATED AT 250 WORDS)

S-100 ANTIGEN-POSITIVE FOLLICULOSTELLATE CELLS ARE NOT THE SOURCE OF IL-6 GENE EXPRESSION IN HUMAN PITUITARY ADENOMAS

We have investigated the expression of IL‐6 in a random selection of 27 human pituitary adenomas, comprising 8 somatotroph, 5 corticotroph, 3 mammotroph and 11 endocrinologically inactive adenomas, using a 35S‐labelled 1.1kb riboprobe complementary to human IL‐6. Positive and negative IL‐6 transcript controls were generated from the IL‐6‐secreting human bladder carcinoma cell line T24/83. Tissue from a malignant melanoma was used as a positive S‐100 immunocytochemical control tissue. Of the 27 human pituitary adenomas examined by in situ hybridization, 7 (26%) contained IL‐6 transcripts: these were 3 of 5 corticotroph adenomas, 2 of 8 somatotrophinomas and 2 of 11 endocrinologically inactive adenomas. In each case, IL‐6 transcript‐positive cells constituted less than 1% of the total pituitary tissue mass examined. Alternate wax embedded 3 μm thick sections from 5 of the 7 IL‐6 transcript positive tumours were examined immunocytochemically for S‐100 antigen, or by in situ hybridization for IL‐6 transcripts. Immunocytochemistry for S‐100 antigen was completely negative in 3 of the 5 tumours and in the remaining 2, there was no evidence of IL‐6 transcripts and S–100 antigen co‐localization in any of the sections examined. This suggests that in pituitary adenomas, cells other than classical folliculostellate cells are responsible for IL‐6 production.

Substance P and substance K in the median eminence and paraventricular nucleus of the rat hypothalamus.

We have used specific radioimmunoassays coupled with reversed-phase high-performance liquid chromatography (HPLC) to measure and characterise substance P (SP) and substance K (SK) in subdivisions of the rat hypothalamus. SP and SK levels in the paraventricular nucleus (PVN) were 968 +/- 61 and 381 +/- 22 pg respectively; in the supraoptic nucleus (SON) were 210 +/- 21 and 79 +/- 8 pg; and in the median eminence/arcuate nucleus (ME) were 1044 +/- 66 and 451 +/- 20 pg. Reversed-phase HPLC revealed that immunoreactive (ir) SP was present solely in the non-oxidised form in all tissue extracts. The principal form of ir-SK in the PVN and SON coeluted with synthetic SK on HPLC, but some immunoreactivity eluted in a later position. This material represented less then 5% of the total ir-SK in extracts of the PVN and SON, but increased to 35-40% of the total in the ME. Gel chromatography and HPLC characterised this compound as being slightly smaller and more hydrophobic than SK. These results establish that ir-SK is present within the hypothalamus in varying amounts and molecular forms. The location of significant amounts of both SP and SK in the PVN and ME, the principal regions of CRF-41 synthesis and release, is compatible with a role for neurokinins in the modulation of CRF-41 and consequently ACTH release.

Substance P and substance K in the rat hypothalamus following monosodium glutamate lesions of the arcuate nucleus.

Adult rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Following MSG lesioning, dopamine content in median eminence/arcuate nucleus (ME/AN) tissue extracts declined by 60-70%. Substance P (SP) content as determined by radioimmunoassay was significantly decreased in the paraventricular nucleus (PVN) (531 +/- 30 pg, mean +/- SEM) compared to controls (871 +/- 110 pg) but was unchanged in ME/AN extracts. Substance K (SK) content decreased to 257 +/- 20 pg in the PVN of lesioned animals compared to controls (367 +/- 31 pg) and the ME/AN content of SK was also significantly decreased (236 +/- 36 pg compared to control levels of 619 +/- 65 pg). The CRF-41 content of the PVN and ME/AN was unchanged by MSG lesioning, indicating that these areas are not affected by MSG. The partial depletion of SP and SK in the PVN following MSG treatment provides evidence that at least some of the neurokinin content of the PVN may originate in cell bodies of the arcuate nucleus. However, the lack of response of ME/AN SP to MSG treatment may suggest that the arcuate nucleus is not the major source of SP in the median eminence.