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Partial cloning of the cDNA encoding the hamster VIP receptor
102 PARTIAL CLONING OF THE cDNA ENCODING THE HAMSTER VIP RECEPTOR
J.V. GARDINER, P.M. JONES, S.R. BLOOM Royal Postgraduate Medical School, Du Cane Rd, LONDON, W12 ONN Vasoactive intestinal polypeptide (VIP) is a member of the secretin/glucagon family of peptides with a wide variety of known physiological roles. One of these functions is as a potent stimulator of insulin secretion in both isolated islets and a hamster insulinoma cell line (HIT T15) used as a model of B-cell function. To clone the receptor for VIP, a polymerase chain reaction (PCR) approach was chosen. The published sequences of several receptors which function by activating adenylate cyclase namely secretin, calcitonin and PTH were aligned and three of the most highly conserved regions were selected to design degenerate primers for use in a semi-nested PCR reaction. HIT T15 cells were used as the source of mRNA for use in the reverse transcription and subsequent amplification. The first round of PCR gave a number of bands when visualised on an agarose gel. When the product of this PCR was subjected to a second round of amplification using an internal primer at the 3' end, the result was a single band of the expected size when visualised on an agarose gel. This fragment was sub-cloned into the plasmid, Bluescript. One insert was found to have a 72% homology to the secretin receptor. This clone was then demonstrated to have 96% homology to the later published rat VIP receptor which suggests that this clone represents part of the hamster VIP receptor. These results demonstrate the use of PCR in the identification and characterisation of G-protein linked receptors and that the VIP receptor shows a high degree of sequence conservation across these two species of mammal
REGULATION OF ANTERIOR PITUITARY GALANIN BY OESTROGEN AND PROLACTIN STATUS PJ HAMMOND, N KHANDAN-NIA, MA GHATEI, SR BLOOM Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 0NN Galanin is an important autocrine regulator of lactotroph function. Anterior pituitary (AP) galanin is very sensitive to the oestrogen status of the animal, hyperoestrogenization increasing levels of galanin mRNA up to 4000-fold, while prolactin is a negative regulator of AP galanin. We investigated the interaction of prolactin and oestrogen in the regulation of AP galanin. To induce hyperprolactinaemia female rats had three anterior pituitary glands implanted under their renal capsules. Five groups of animals were studied: control; hypophysectomized with steroid and thyroxine replacement and AP implants; AP implants; hyperoestrogenization; hyperoestrogenization and AP implants. Hypophysectomized animals with AP implants had similar circulating prolactin levels to control animals (45.5 +4.45 vs. 56.43 +7.87 ng/ml) but had no detectable galanin content. AP implants in control animals increased circulating prolactin levels (94.6+11.99 ng/ml, p<0.01) and this resulted in a decrease in AP galanin (21.3+7.26 vs. 58.8+8.37 fmol/#g, p<0.01). Galanin was detectable in the implants (4.36+ 1.12 fmol//zg). Hyperoestrogenization increased circulating prolactin levels significantly more than the implants (141.9+13.4 ng/ml, p<0.000 vs.control, p<0.01 vs. implant) and caused a massive increase in AP galanin (1168+163 , p<0.000). AP implants did not significantly augment the oestrogen induced hyperprolactinaemia (159+8.6 ng/ml) but did reduce the increase in AP galanin (844.8+91 fmol//~g, p <0.05 vs. hyperoestrogenization). Thus oestrogen status and prolactin status interact with opposite effects in the regulation of AP galanin.
J.V. GARDINER, P.M. JONES, S.R. BLOOM Royal Postgraduate Medical School, Du Cane Rd, LONDON, W12 ONN Vasoactive intestinal polypeptide (VIP) is a member of the secretin/glucagon family of peptides with a wide variety of known physiological roles. One of these functions is as a potent stimulator of insulin secretion in both isolated islets and a hamster insulinoma cell line (HIT T15) used as a model of B-cell function. To clone the receptor for VIP, a polymerase chain reaction (PCR) approach was chosen. The published sequences of several receptors which function by activating adenylate cyclase namely secretin, calcitonin and PTH were aligned and three of the most highly conserved regions were selected to design degenerate primers for use in a semi-nested PCR reaction. HIT T15 cells were used as the source of mRNA for use in the reverse transcription and subsequent amplification. The first round of PCR gave a number of bands when visualised on an agarose gel. When the product of this PCR was subjected to a second round of amplification using an internal primer at the 3' end, the result was a single band of the expected size when visualised on an agarose gel. This fragment was sub-cloned into the plasmid, Bluescript. One insert was found to have a 72% homology to the secretin receptor. This clone was then demonstrated to have 96% homology to the later published rat VIP receptor which suggests that this clone represents part of the hamster VIP receptor. These results demonstrate the use of PCR in the identification and characterisation of G-protein linked receptors and that the VIP receptor shows a high degree of sequence conservation across these two species of mammal
REGULATION OF ANTERIOR PITUITARY GALANIN BY OESTROGEN AND PROLACTIN STATUS PJ HAMMOND, N KHANDAN-NIA, MA GHATEI, SR BLOOM Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 0NN Galanin is an important autocrine regulator of lactotroph function. Anterior pituitary (AP) galanin is very sensitive to the oestrogen status of the animal, hyperoestrogenization increasing levels of galanin mRNA up to 4000-fold, while prolactin is a negative regulator of AP galanin. We investigated the interaction of prolactin and oestrogen in the regulation of AP galanin. To induce hyperprolactinaemia female rats had three anterior pituitary glands implanted under their renal capsules. Five groups of animals were studied: control; hypophysectomized with steroid and thyroxine replacement and AP implants; AP implants; hyperoestrogenization; hyperoestrogenization and AP implants. Hypophysectomized animals with AP implants had similar circulating prolactin levels to control animals (45.5 +4.45 vs. 56.43 +7.87 ng/ml) but had no detectable galanin content. AP implants in control animals increased circulating prolactin levels (94.6+11.99 ng/ml, p<0.01) and this resulted in a decrease in AP galanin (21.3+7.26 vs. 58.8+8.37 fmol/#g, p<0.01). Galanin was detectable in the implants (4.36+ 1.12 fmol//zg). Hyperoestrogenization increased circulating prolactin levels significantly more than the implants (141.9+13.4 ng/ml, p<0.000 vs.control, p<0.01 vs. implant) and caused a massive increase in AP galanin (1168+163 , p<0.000). AP implants did not significantly augment the oestrogen induced hyperprolactinaemia (159+8.6 ng/ml) but did reduce the increase in AP galanin (844.8+91 fmol//~g, p <0.05 vs. hyperoestrogenization). Thus oestrogen status and prolactin status interact with opposite effects in the regulation of AP galanin.