Intracorporeal Videoprobe (IVP).

The objective of the work in the EC project IVP is the development and evaluation of two prototypes of video systems:- a small wired videoprobe with a CMOS image sensorand- an autonomous video-capsule with a telemetric link for image data transfer to an external PC-based system.

Lactation abolishes corticotropin-releasing factor-induced oxytocin secretion in the conscious rat.

We have assessed the response of plasma oxytocin (OT) to intracerebroventricular CRF-41 in both virgin female and lactating rats. In virgin rats CRF-41 resulted in an increase in plasma OT from 5-30 min after administration. In lactating rats, however, there was a complete abolition of the OT response, even at the highest dose of CRF-41. These data demonstrate another feature of the hormone nonresponsiveness apparent during lactation and suggests that one of the reasons for the lack of stress responses could be a down-regulation of the response to endogenously released CRF-41.

Hypothalamic nitric oxide synthase gene expression is regulated by thyroid hormones.

We investigated the effects of thyroid status on nitric oxide synthase (NOS) gene expression in the rat hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). Propylthiouracil (PTU)-induced hypothyroidism in male rats produced a highly significant reduction in NOS gene transcripts in the PVN and SON, as assessed by quantitative in situ hybridization histochemistry with a specific oligodeoxynucleotide probe. The addition of T3 (40 micrograms/kg) to the PTU-containing diet completely prevented the reduction in NOS transcripts. Hyperthyroidism, induced by adding 160 micrograms/kg T3 to the food, more than doubled the prevalence of NOS transcripts in the PVN and SON after a similar time. Up-regulation of NOS gene transcripts induced by the osmotic stimulus of chronic salt loading was markedly attenuated by PTU-induced hypothyroidism. These results demonstrate a major effect of thyroid status on regulation of NOS gene expression in the hypothalamus.

Chronic activation of the hypothalamo-pituitary-adrenal axis and loss of circadian rhythm during adjuvant-induced arthritis in the rat.

Adjuvant-induced arthritis (AA) in Sprague-Dawley rats resulted in a chronic increase in plasma levels of ACTH and corticosterone (B). Joint inflammation became clinically apparent between days 12-15 after injection of adjuvant and reached a peak on day 21, after which time it subsided. In AA animals, plasma ACTH and B levels in the morning (0800-0900 h) on days 7, 8, 9, and 21 were significantly higher than those in control animals (day 0). The corresponding evening ACTH and B levels in AA animals were not significantly different from evening levels in the control animals. Adrenal weight in AA animals was increased on day 21, while thymic weight diminished gradually from days 7-21 postinjection. Development of AA was associated with activation of the hypothalamo-pituitary-adrenal axis, with increased morning ACTH and B levels and abolition of normal diurnal ACTH and B rhythms. This model of chronic inflammatory stress clearly activates the ACTH drive despite increased corticosteroid feedback in the morning, resulting overall in chronically increased B secretion.

Neuropeptide-Y potentiates the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland.

Neuropeptide-Y (NPY) is colocalized with vasopressin and oxytocin in magnocellular neurons of the hypothalamo-neurohypophysial system, and a very high density of NPY-binding sites is present within the neurohypophysis. To investigate the possibility that NPY exerts a modulatory role on the release of neurohypophysial hormones, we have studied the actions of NPY on potassium-evoked release of vasopressin and oxytocin from the rat neurointermediate lobe in vitro. NPY dose-dependently potentiated vasopressin release evoked by high extracellular potassium (56 mM), with a maximal enhancement of 223% (10(-7) M NPY). A similar effect was obtained with the Y2-selective agonist NPY-(13-36). In contrast, no effect on the potassium-evoked release of oxytocin was observed at this concentration. In the absence of Ca2+ in the incubation medium, NPY did not potentiate vasopressin secretion, indicating that the effect of NPY on potassium-evoked secretion of neurohypophysial vasopressin is critically dependent on extracellular calcium ions. The number of neurohypophysial NPY-binding sites is drastically down-regulated in animals subjected to chronic osmotic stimulation. In the present study, it was observed that the potentiating effect of NPY on vasopressin secretion was completely abolished in neurointermediate lobes recovered from animals that had been drinking 2% NaCl for 12 days, reflecting the concomitant down-regulation of neurohypophysial NPY-binding sites observed during this state. Finally, it was confirmed that stimulation by high K+ significantly evoked the release of endogenous NPY from neurointermediate lobes of the pituitary gland. The present results provide evidence that NPY selectively and potently enhances evoked vasopressin secretion. Considering the coexistence of the two neuropeptides in magnocellular hypothalamo-neurohypophysial neurons, this action is likely to be part of an autostimulatory feedforward loop. NPY may be an important component in the mechanisms associated with the control of body fluid homeostasis.

Glucocorticoid-mediated responses of plasma ACTH and anterior pituitary pro-opiomelanocortin, growth hormone and prolactin mRNAs during adjuvant-induced arthritis in the rat.

Adjuvant arthritis (AA) in the rat leads to chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and the loss of its diurnal rhythmicity. We have investigated the effects of adrenalectomy (ADX) and different levels of corticosterone replacement upon plasma ACTH levels and anterior pituitary pro-opiomelanocortin (POMC), GH and prolactin mRNAs during the development of AA. In control ADX animals, we observed the negative feedback effects of exogenous corticosterone on plasma ACTH and anterior pituitary POMC mRNA. In the ADX animal with AA, however, the increased POMC mRNA which was observed was not reduced by exogenous corticosterone on day 7 of AA, although the negative feedback effect of corticosterone on plasma ACTH was intact. On day 14, however, even high dose corticosterone replacement failed to have a significant feedback effect on the raised levels of plasma ACTH. In control ADX animals, corticosterone replacement resulted in increased anterior pituitary GH mRNA and reduced prolactin mRNA. In contrast, in ADX animals with AA, GH mRNA was reduced and there was a further decrease in prolactin mRNA. In these animals, corticosterone replacement did not affect GH or prolactin mRNA expression. These data demonstrate a disruption of the normal mechanisms underlying feedback inhibition of the HPA axis by glucocorticoids during AA. Similarly, the glucocorticoid-dependent regulation of GH and prolactin mRNA expression is altered in AA.

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)

Vasopressin is located within lymphocytes in the rat spleen.

Using dual antigen immunocytochemical staining with a specific antiserum for arginine vasopressin (AVP), we have detected AVP immunoreactivity in clusters of large immunoglobulin (Ig) G-containing cells, probably plasma cells, within the rat spleen, and in smaller cells which are IgG-negative. Vasopressin-positive cells were detected principally throughout the white pulp areas in the subcortical region of the spleen. IgG staining could only be detected within the cells and not on the cell surface, demonstrating that the antiserum is recognising genuine intracellular IgG and not cell surface antigens. Reversed-phase HPLC of spleen tissue extract revealed a single peak of AVP immunoreactivity which co-eluted with the standard. This is the first evidence that AVP is found within lymphocytes of the immune system and provides further information about the important interaction between the endocrine and immune systems.

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)

Response of pituitary and spleen pro-opiomelanocortin mRNA, and spleen and thymus interleukin-1 beta mRNA to adjuvant arthritis in the rat.

During development of adjuvant-induced arthritis (AA) in the rat, pituitary pro-opiomelanocortin (POMC) mRNA expression was increased. Pituitary POMC mRNA was much higher following adrenalectomy and AA. Spleen POMC mRNA also increased with a similar time kinetics, although the levels in the spleen were much lower than those in the pituitary. In control animals, spleen interleukin-1 beta (IL-1 beta mRNA) was undetectable, whereas AA led to the accumulation of IL-1 beta mRNA and the highest levels were seen in the adrenalectomised AA group. Thymic IL-1 beta expression was also increased in AA animals. These results suggest that AA leads to the activation of both the neuroendocrine and the immune systems and the interaction between these systems may play a role in this disease state.

Effects of cyclosporine A on the hypothalamic-pituitary-adrenal axis and anterior pituitary interleukin-6 mRNA expression during chronic inflammatory stress in the rat.

In the rat, adjuvant arthritis (AA) is an inflammatory joint disease associated with chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. We have investigated the effects of the immunosuppressive agent cyclosporine A (CsA) on plasma levels of adrenocorticotropin (ACTH) and corticosterone (B), as well as on anterior pituitary proopiomelanocortin (POMC) and interleukin (IL)-6 mRNA accumulation in control and adjuvant-injected animals. In control animals, CsA reduced basal anterior pituitary POMC and IL-6 mRNA and decreased plasma levels of ACTH and B. Adjuvant-injected animals that were treated with CsA showed no clinical signs of AA. Moreover, CsA inhibited the arthritis-induced increases in pituitary POMC and IL-6 mRNA levels and in circulating ACTH and B. In vitro, CsA reduced the POMC mRNA content of cultured anterior pituitary cells and diminished the stimulatory effects of corticotropin-releasing hormone (CRH) on POMC mRNA expression and ACTH secretion from these cells. These data indicate that CsA has a direct action on the HPA axis and also reduces the activation of the HPA axis seen in chronic inflammatory arthritis.

Central inhibition by gamma-aminobutyric acid of the release of vasopressin by carbachol in the rat.

1. gamma-Aminobutyric acid (GABA) inhibited the antidiuretic response and the increased urinary excretion of vasopressin produced by carbachol when both drugs were injected into a lateral cerebral ventricle (i.c.v.) in the water-loaded rat under ethanol anaesthesia. 2. The inhibitory effect of GABA was mimicked by muscimol and 3-amino-1-propane sulphonic acid (3-APS) and blocked by bicuculline. 3. GABA injected i.v. or into the cisterna magna (i.cist.) did not inhibit the release of vasopressin by carbachol injected i.c.v. 4. The results suggest a role for GABA as a putative inhibitory transmitter in the hypothalamo-neurohypophysial system, acting directly on the supraoptic or paraventricular nuclei in the anterior hypothalamus.

Release of vasopressin by enkephalin.

Leu-enkephalin, its stable analogue [D-Ala2-D-Leu5]-enkephalin and the C-fragment of lipotropin (beta endorphin) injected intravenously in the rat produced antidiuretic responses which were inhibited reversibly by naloxone. It was shown for Leu-enkephalin that injection into the cerebral ventricles was at least ten times more effective than intravenous injection and for [D-Ala2-D-Leu5]-enkephalin that the antidiuretic response was associated with increased excretion of vaspressin in the urine.

Hydroxy analogues of oxytocin and of lysine-vasopressin.

1 Synthetic analogues of oxytocin and of lysine-vasopressin with an hydroxyl group in either the L ro D configuration replacing the primary amino group have been tested for biological activity.2 [1-(L-2-Hydroxy-3-mercaptopropanoic acid)] oxytocin ([L-Hmp(1)]oxytocin) was 1.5 to 2 times more potent than oxytocin on the rat uterus in situ, the rat mammary strip and the rat mammary gland in situ and 3 times more potent on the rat isolated uterus.3 The pressor activity of [1-(L-2-hydroxy-3-mercaptopropanoic acid)-8-lysine]vasopressin ([L-Hmp(1), Lys(8)] vasopressin) was 2.2 and the antidiuretic activity 2.1 times that of lysine-vasopressin.4 The [D-Hmp(1)] analogues of oxytocin and vasopressin were much less potent than the [L-Hmp(1)] analogues.5 The responses to oxytocin and its hydroxy analogues in vivo were qualitatively indistinguishable but the pressor and antidiuretic responses to the hydroxy analogues of lysine-vasopressin were prolonged compared with those to the parent hormone.6 The hydroxy analogues of oxytocin and lysine-vasopressin were not inactivated by pregnancy plasma oxytocinase.7 The results are discussed in relation to the importance of the primary amino group for the biological activity and metabolism of the neurohypophysial hormones.

Central inhibition by gamma-aminobutyric acid and muscimol of the release of vasopressin and oxytocin by an osmotic stimulus in the rat.

1. In water-loaded rats under ethanol anaesthesia, the injection of 2-4 microliters 1.54M NaCl solution (hypertonic saline:HS) into a lateral cerebral ventricle (i.c.v.) produced an antidiuretic and a pressor response, together with increased urinary excretion of vasopressin and 'oxytocin-like radioimmunoreactivity' (OLRI). In lactating rats HS also produced a milk-ejection response which was shown to be due to the release of oxytocin. 2. The injection of 20-40 micrograms gamma-aminobutyric acid (GABA) or 40-80 ng muscimol i.c.v. 2 min before HS inhibited the antidiuretic, pressor and milk-ejection responses and reduced the urinary excretion of vasopressin and OLRI. 3. The pressor response to HS was abolished by a ganglion blocking agent but it was not reduced by a vasopressin antagonist. After the antagonist, the antidiuretic response to HS was abolished and the pressor response was accompanied by a diuresis both of which were blocked by muscimol. 4. The threshold dose of HS for an antidiuretic response was 4-8 times higher on injection into the cisterna magna (i.cist.) than when injected i.c.v. GABA, i.v. or i.cist, did not inhibit the response to HS i.c.v. 5. The results confirm other evidence that, in the rat, in contrast some other species, an osmotic stimulus causes release of both vasopressin and oxytocin. This release is blocked by GABA and muscimol. These drugs and HS act at a site reached not from the subarachnoid space but from the cerebral ventricles, probably the hypothalamus. The pressor response to HS under the experimental conditions used is due entirely to central sympathetic stimulation and this effect, as well as the release of vasopressin and oxytocin, is blocked by muscimol.

Evidence for arginine vasopressin as the primary activator of the HPA axis during adjuvant-induced arthritis.

1. Adjuvant-induced arthritis (AA) is an experimental inflammation of the joints that results in chronic activation of the hypothalamo-pituitary-adrenal (HPA) axis. 2. In this study the role of hypothalamic corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of the HPA axis in this condition both in Sprague-Dawley (SD), and Piebald-Viral-Glaxo (PVG) rats has been further characterized. 3. The increase in AVP peptide content of portal blood (as early as day 11), just prior to the onset of arthritis is confirmed and further increases, peaking at day 16 are shown, coincident with the progression of inflammation in the PVG rats. 4. The increase in AVP is associated with a significant increase in the expression of AVP but not CRF mRNAs in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN) of arthritic SD rats. 5. In the presence of maximal inflammation of SD rats there was a significant decrease in the maximum binding of [125I]-Tyr-oCRF to anterior pituitary membranes, whereas AVP receptor concentration in anterior pituitary membranes from both PVG and SD rats showed a significant increase with respect to controls. 6. The basal adrenocorticotrophin (ACTH) secretion in vitro was similar in both control and arthritic SD rats but that from arthritic PVG rat pituitaries was significantly greater than the respective controls (436 +/- 91 v 167 +/- 23 pg/tube). The ACTH response of pituitaries of arthritic PVG rats to CRF or the combination of CRF and AVP was significantly higher compared with the controls, although the ACTH response of arthritic SD rat pituitaries was unchanged. 7. The results are consistent with the view that activation of the parvocellular vasopressin system has an important role in the adaptation of the HPA axis to experimentally-induced chronic stress of arthritis.

Neonatal monosodium glutamate treatment alters both the activity and the sensitivity of the rat hypothalamo-pituitary-adrenocortical axis.

We have investigated the effects of monosodium glutamate (MSG) lesioning of the arcuate nucleus on both central and peripheral components of the hypothalamo-pituitary-adrenocortical (HPA) axis under basal conditions and under acute and chronic stress. Plasma ACTH levels were lower in MSG-lesioned rats (27 +/- 7 pg/ml) compared with controls (71 +/- 18 pg/ml) while corticosterone levels were elevated (523 +/- 84 ng/ml compared with 176 +/- 34 ng/ml). Quantitative in situ hybridization histochemistry revealed that corticotrophin-releasing factor mRNA levels in the medial parvocellular part of the hypothalamic paraventricular nucleus were significantly lower in MSG-treated rats. MSG lesioning resulted in an enhanced response of corticosterone to restraint stress (1309 +/- 92 ng/ml compared with 628 +/- 125 ng/ml in sham-lesioned animals), while ACTH responses to restraint stress in MSG-lesioned and sham-MSG groups were not significantly different (160 +/- 24 pg/ml and 167 +/- 24 pg/ml respectively). These data suggest that MSG-lesioned rats have an increased adrenocortical sensitivity. In rats subjected to the chronic osmotic stimulus of drinking 2% saline for 12 days, plasma ACTH levels were significantly reduced (15 +/- 5 pg/ml) and the ACTH and corticosterone responses to restraint stress were eliminated. ACTH levels were also reduced in MSG-treated animals given 2% saline and the ACTH response to acute stress remained absent in these animals. However, a robust corticosterone response to restraint stress was observed in saline-treated MSG-lesioned rats.(ABSTRACT TRUNCATED AT 250 WORDS)

C/EBP activates the human corticotropin-releasing hormone gene promoter.

The purpose of these studies was to identify whether transcription factors, associated with cytokine signalling, affected promoter activity of the corticotropin releasing hormone (CRH) gene. Fragments of a 3.6 kb sequence of the human CRH gene promoter were amplified by PCR and ligated upstream of a CAT reporter. These constructs were transfected into a variety of cell lines, either alone or together, with transcription factor expression vectors. Basal activity of a 3070 bp CRH promoter fragment was only seen in neuronal and lymphoblastoid cell lines. Promoter activity was increased by the transcription factors C/EBPbeta (NF-IL6) and more strongly, by C/EBPdelta (NF-IL6beta). Increased CRH promoter activity following phorbol ester treatment was inhibited by a dominant negative NF-IL6 mutant, showing that the effects of phorbol ester were principally mediated by C/EBP. Moreover, the inverse changes in the expression of CRH in the hypothalamus and spleens of arthritic rats were paralleled by similar inverse changes in NF-IL6beta expression in these organs. These data show that some transcription factors associated with cytokine signalling can also activate the CRH promoter.

Pharmacokinetics and efficacy of the long-acting somatostatin analogue somatuline in acromegaly.

The aim of this work was to assess the use of a sustained-release formulation of somatuline, a long-acting analogue of somatostatin, in the treatment of acromegaly. Fifteen patients with active acromegaly, as defined by random growth hormone (GH) levels greater than 10 mU/l, which fail to be suppressed to less than 5 mU/l following an oral glucose load, were studied. Somatuline was administered as an intramuscular injection in two regimens: eight patients were given a single injection of the sustained-release formulation and blood samples taken over the next month for the measurement of both basal levels of GH and the GH response to thyrotrophin-releasing hormone; and eight patients were given injections of the sustained-release formulation at 2-week intervals over a 6-month period and basal plasma GH levels and the GH response to both an oral glucose load and to thyrotrophin-releasing hormone was assessed. Following a single intramuscular dose of the sustained-release preparation, random GH levels were reduced to below 10 mU/l in five patients and by greater than 50% of basal levels in the remainder. The insulin-like growth factor I (IGF-I) levels fell to within the normal range in three patients. In the long-term efficacy study. GH levels were reduced to < 10 mU/l in 7/8 patients. The IGF-I levels were normalized in four patients. Five of the eight patients experienced diarrhoea, two of mild and three of moderate severity; none of the patients withdrew from the study.(ABSTRACT TRUNCATED AT 250 WORDS)

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.