Abstract

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal β-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, 03, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7·5 μm, compared with a scrambled (MS03) control oligo (n=3, P<0·01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1·78 ± 0·13 (MS03) vs 0·19 ± 0·14 (03) pmol/10⁶ cells per 240 min, n=7, P<0·01). A significant increase in insulin mRNA (100 ± 10% (MS03) vs 124 ± 8% (03), n=3, P<0·05) and insulin content (13·0 ± 0·9 (MS03) vs 17·4 ± 1·4 (03) pmol/10⁶ cells, n=7, P=0·028) was observed following treatment with 03 at 7·5 μm. 08, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than 03, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated β-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.

Journal of Endocrinology (1996) 151, 341–348

ABSTRACT

Neuropeptide Y (NPY), neurotensin (NT), substance P (SP) and vasoactive intestinal peptide (VIP) are four structurally unrelated neuroendocrine peptides which affect anterior pituitary function. All four peptides appear to be locally synthesized in the anterior pituitary gland and have been shown to be regulated by thyroid and/or sex hormone status. We show here that NT, SP and VIP but not NPY are influenced by adrenal hormone status in the male rat pituitary gland. Adrenalectomy increased the content of VIP (35·4±4·0 (s.e.m.) vs control 11·9±1·1 pmol/g wet weight) but decreased that of SP (18·8±2·3 vs control 36·7 ±3·9 pmol/g wet weight). Adrenalectomy combined with castration decreased the content of SP (14·6±3·5 vs control 36·7±3·9 pmol/g wet weight) but had no effect on VIP content. Treatment with dexamethasone produced significant decreases in NT, SP and VIP contents (17·8±2·3 vs control 32·6±3·4 pmol/g wet weight, 5·5±0·9 vs control 36·7±3·9 pmol/g wet weight and 4·2±0·6 vs control 11·9±1·1 pmol/g wet weight respectively). The changes in pituitary peptide contents occurred in parallel with changes in mRNA levels, suggesting that alterations in glucocorticoid hormone status can alter the synthesis of these peptides. These results, together with the known effects of these neuroendocrine peptides suggest possible functions for locally produced SP and VIP in regulating the secretion of adrenocorticotrophin and/or other proopiomelanocortin-derived peptides. The differential effects of adrenalectomy and adrenalectomy combined with castration on VIP suggests that the regulation of this pituitary peptide is complex, and may be dependent, in part at least, on gonadal factors.

Journal of Endocrinology (1990) 127, 437–444

ABSTRACT

Galanin-like immunoreactivity (IR) was measured by radioimmunoassay in extracts of non-tumorous and tumorous human pituitaries and in multiple sites in the human brain. Galanin-IR was present in considerable quantities in the non-tumorous pituitaries (21·4±1·2 pmol/g wet weight; mean ± s.e.m., n = 30). In 25 pituitary tumours, galanin-IR was detectable in extracts of only nine, with a mean concentration of 11·5±4·4 pmol/g. Galanin-IR was undetectable in the remaining 16. Of ten brain sites, galanin-IR was detected only in the hypothalamus, where the concentration was 9·1±1·8 pmol/g (n = 5). On fast protein liquid chromatography of the non-tumorous pituitary extracts, galanin-IR mostly eluted in a peak with a retention time similar to that of synthetic porcine galanin. On gel permeation chromatography, galanin-IR eluted as a peak with an elution coefficient (K _av) of 0·72, also similar to that of porcine galanin, with additional preceding (K _av 0·62) and following (K _av 0·77) peaks of galanin-IR. These results show that healthy human pituitary and hypothalamus contain substantial amounts of galanin, whereas it is present in variable amounts or not at all in pituitary tumours. Chromatographic analysis suggests that pituitary galanin is present in three molecular forms, with the majority corresponding to synthetic porcine galanin.

Journal of Endocrinology (1991) 130, 463–467

ABSTRACT

Prolactin secretion is highly regulable, and the possibility exists that there are local intrapituitary factors controlling prolactin secretion. Recently, the neuropeptides vasoactive intestinal peptide (VIP), galanin and substance P (SP) have been co-localized to the lactotroph in the female rat. We investigated the effects of alterations in prolactin status in vivo on pituitary and hypothalamic expression of these peptides by specific radioimmunoassays and mRNA analysis. In the anterior pituitary, following haloperidol treatment, the contents of both VIP and galanin were suppressed to below detectable levels. Similarly, after bromocriptine treatment, the content of VIP was decreased to below the detection limit of the assay while galanin (14·2±1·3 vs control 21·0±2·1 fmol/mg, P < 0·05) also showed a significant reduction. The levels of VIP mRNA and galanin mRNA in these groups showed the same qualitative change as their respective peptides. Concurrent treatment with highdose oestrogen modified the VIP peptide response to bromocriptine (1368·7 ±149·2 vs bromocriptine 843·4±82·7 fmol/mg, P<0·05) but not to haloperidol. Oestrogen-induced decreases in galanin content were not influenced by either treatment. The pituitary content of SP showed a fall after oestrogen treatment (1·1±0·01 vs control 6·4±0·8 fmol/mg, P<0·05) which was not significantly altered by either bromocriptine or haloperidol. Likewise, SP mRNA levels in the pituitary were decreased by 90% following oestrogen treatment. Hypothalamic expression of these peptides did not change with any of the treatments. This study demonstrates the tissue and peptide specific influences of prolactin status in regulating anterior pituitary VIP, galanin and SP at the pretranslational level. The results of this study suggest that prolactin status and/or dopaminergic influences are important in the regulation of anterior pituitary neuropeptides.

Journal of Endocrinology (1991) 131, 411–419

ABSTRACT

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalami and so called because of its ability to stimulate pituitary adenylate cyclase activity. Alternative amidation and proteolytic processing of prepro-PACAP gives rise to two bioactive-amidated forms, PACAP-NH₂(1–38) (PACAP-38) and PACAP-NH₂(1–27) (PACAP-27). 7B2 is a polypeptide of 185 amino acids which is predominantly found in secretory granules and is widely distributed in rat and human tissues. We investigated the ability of the two forms of PACAP to stimulate GH, prolactin and 7B2 release by the rat pituitary clonal cell line GH₃, and ACTH and 7B2 by the mouse pituitary clonal cell line AtT-20. PACAP-38 and PACAP-27 stimulated 7B2 and GH/prolactin or ACTH secretion with a similar efficacy over the 2-h incubation period from GH₃ and AtT-20 cells respectively. 7B2 secretion was also stimulated by corticotrophin-releasing factor (CRF-41) and vasoactive intestinal polypeptide (VIP) in AtT-20 cells, and thyrotrophin-releasing hormone (TRH) and VIP in GH₃ cells. Addition of PACAP to CRF-41 resulted in an additive effect on ACTH secretion and a synergistic effect on 7B2 secretion in AtT-20 cells. No synergism was observed when PACAP was added together with TRH, either on GH and prolactin secretion or on 7B2 release from GH3 cells. PACAP-mediated 7B2 secretion from both cell lines and PACAP-stimulated ACTH release from AtT-20 cells were reduced by 5 mg octapeptide synthetic somatostatin analogue/l (5 mg SMS 201-995/1).

Journal of Endocrinology (1992) 132, 107–113

Abstract

Insulin secretion is regulated by neural and neurohormonal factors. The report of nerves releasing pituitary adenylate cyclase-activating polypeptide (PACAP) – a 38 amino acid peptide – in the endocrine pancreas, suggests it may be important in modulating insulin release. We therefore carried out receptor-binding studies on membranes prepared from the glucose-responsive clonal β-cell line HIT-T15, and also examined the effects of PACAP38, PACAP27 – a C-terminal truncated form of the peptide – and vasoactive intestinal peptide (VIP) on insulin and islet amyloid polypeptide (IAPP) release. We showed by chemical cross-linking that PACAP and VIP stimulate secretion from the clonal cells by binding to a receptor with a molecular weight of 67 kDa (n=4). Binding was saturable when membranes were incubated with ¹²⁵I-PACAP27 (K _d 1·2±0·2 nm; B_max 415·7±35·3 fmol/mg; n=4) or ¹²⁵I-VIP (K _d 1·3±0·3 nm; B_max 354·8 ± 42·8 fmol/mg; n=4). We also demonstrated an increase in glucose-stimulated insulin (PACAP27, 366·6 ±25·8% PACAP38, 389·9 ±13·4%; VIP, 342·6± 16·1% of control; all at 1 μm, P<0·01 vs control) and IAPP release (PACAP27, 236·9 ±26·2%; PACAP38, 226·5 ± 10·9%; VIP, 242·9 ± 15·8% of control; all at 1 μm, P<0·01 vs control). Incubation of the cells with these peptides, for a duration of 12 h, in the presence of 5·5 mm glucose, did not alter the expression of insulin or IAPP. These findings suggest that PACAP and VIP stimulate secretion of insulin and IAPP by binding to a 67 kDa protein on clonal β-cells and do not alter the transcription of insulin and IAPP under the conditions tested.

Journal of Endocrinology (1995) 147, 121–130

Abstract

We have compared the effects of thyroidectomy with those of thyroxine (T₄) replacement and excess T₄ treatment on neuropeptide Y (NPY) in the rat anterior pituitary, and compared these with the effects on substance P (SP) and vasoactive intestinal peptide (VIP). Thyroidectomy produced large increases in the peptide content of NPY (335 ± 58 fmol/gland vs 15 ± 4 fmol/gland in controls), SP (581 ±90 vs 199 ±32 fmol/gland) and VIP (1386 ± 395 vs 417 ± 77 fmol/gland) together with large increases in the respective prohormone encoding mRNAs, NPY 21 760%±1290%, preprotachykinin-A (PPT-A; which encodes the substance P precursor) 1744%± 190% and VIP 680% ± 129%. Thyroidectomy together with T₄ replacement produced an increase in both NPY peptide (426 ±72 vs 15 ±4 fmol/gland) and mRNA content 970%±156% of controls). The peptide contents of SP and VIP were not significantly different from controls. PPT-A and VIP mRNA levels were decreased relative to controls (31%±8% and 23%± 10% respectively). In intact animals treated with excess T₄ (hyperthyroid animals), SP and VIP peptide contents were significantly reduced (55 ±13 vs 199±32 fmol/gland and 226 ± 24 vs 417± fmol/gland respectively) and the SP and VIP encoding mRNAs were also decreased (8% ±3% and 11%±4% respectively). In this group there was no detectable alteration in either the peptide or mRNA content of NPY. Thus, the response of pituitary NPY to thyroid hormone manipulations cannot be explained in terms of negative feedback physiology and is different from those of SP and VIP. The results suggest that the regulation of locally produced NPY in the rat anterior pituitary is complex and may be influenced by thyroidal factors in addition to T₄.

Journal of Endocrinology (1994) 143, 393–397

ABSTRACT

The distribution of a novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), was studied in the brain of the rat and man and a variety of other rat tissues using Northern blot hybridization and two radioimmunoassays for PACAP 1–38 and PACAP 1–27. The assay, using PACAP 1–38 as standard and an antibody to PACAP 21–38 and radiolabelled tracer, revealed immunoreactive PACAP in all brain regions examined, with the highest concentrations in the rat being in the hypothalamus, nucleus accumbens and substantia nigra (380 ± 34, 310 ± 37 and 346 ± 30 pmol/g wet tissue, means±s.e.m., n = 5 respectively), whilst in man the highest concentrations were found in the pituitary gland (15·8 ± 4·7 pmol/g). Immunoreactive PACAP 1–38 was also detected in the rat gastrointestinal tract, adrenal gland and testis. The assay using PACAP 1–27 as standard and label and an antibody to PACAP 1–27 detected immunoreactive PACAP only in the rat hypothalamus (12·6 ± 1·8 pmol/g wet tissue, n = 5). PACAP mRNA of approximately 2·7 kb in size was detectable in all brain regions of both rat and man, and its distribution paralleled that of the immunoreactive peptide.

Gel permeation chromatography of different regions of human and rat hypothalamus, and also rat spinal cord and small intestine, showed a broad immunoreactive peak corresponding to PACAP 1–38. Fast protein liquid chromatography (FPLC) resolved this peak into two immunoreactive peaks, the majority eluting in the position of synthetic PACAP 1–38. Presence of immunoreactivity corresponding to PACAP 1–27 was also confirmed in rat hypothalamic extracts using FPLC. The presence of immunoreactive PACAP and its precursor encoding mRNA in various neural and other tissues is in accord with a role for PACAP as a neurotransmitter, neuromodulator or neurohormone.

Journal of Endocrinology (1993) 136, 159–166

ABSTRACT

The N-terminal fragment (PACAP 27) of the novel neuropeptide, pituitary adenylate cyclase-activating polypeptide 38 (PACAP 38), has 68% homology with vasoactive intestinal polypeptide (VIP). The administration of bolus doses of PACAP 38 and its 27 amino acid N-terminal fragment (PACAP 27) caused a rapid but transient dose-dependent hypotensive effect in the anaesthetized rat. The amplitude and duration of the response obtained by PACAP 38 was comparable with VIP whereas PACAP 27 was three times less potent than VIP. Furthermore, radioreceptor binding studies demonstrated that ¹²⁵I-labelled PACAP 27 and ¹²⁵I-labelled VIP bound to membranes prepared from blood vessels. Both PACAP 27 and VIP were capable of displacing the other from these binding sites. We propose that the hypotensive effect is via the same receptor type.

Journal of Endocrinology (1991) 129, 69–73

ABSTRACT

Glucagon-like peptide (GLP)-1 (7–36)-NH₂ is a peptide found in the mucosal endocrine cells of the intestine, and plasma levels of GLP-1 (7–36)-NH₂ immunoreactivity show a rise after the ingestion of a fat or mixed-component meal. We investigated the effects of physiological infusion of GLP-1 (7–36)-NH₂ on a submaximal gastric acid secretion in healthy volunteers at a rate known to mimic the observed postprandial rise in plasma concentrations. Corrected gastric acid output decreased to less than 50% and volume output to 33% of stimulated values. After the infusion, the secretion of gastric acid recovered immediately to preinhibition values. These results suggest a novel role for GLP-1 (7–36)-NH₂ as a physiological inhibitor of gastric acid secretion in man.

Journal of Endocrinology (1990) 126, 169–173