Production of hormones in the ovary is controlled by endocrine, paracrine, autocrine and intracrine influences. Similar controls may exist in the placenta. I wished to investigate the involvement of second messengers in the action of hormones in control of hormonogenesis in rat ovary and human placenta. The second messengers involved in the action of gonadotropin-releasing hormone (GnRH) and prostaglandin (PG) F₂[formula omitted] were investigated in rat granulosa and luteal cells. As well, the endocrine role of GnRH in the placenta and the possible second messengers involved were investigated. Monolayer cultures of rat granulosa and luteal cells and human placental cells were prepared. Rat granulosa cells were mechanically dispersed; rat luteal cells were enzymatically dispersed with collagenase and DNase. Rat granulosa cells were treated during the first 24 hours in culture; rat luteal cells were treated up to 3 days after dispersion. Radioimmunoassay of medium was used to determine the effect of treatments on hormone production. Studies which examined the effect of hormones on the intracellular free calcium concentration ([Ca²⁺]i) in single cells using the calcium sensitive fluorescent dye, Fura-2, were done in monolayer rat granulosa and luteal cell cultures. Human placental cells, from first trimester and term placentae, were dispersed using trypsin-DNase or collagenase-DNase. Cells were cultured for 2 days prior to treatment. The effects of treatments on production of steroid (progesterone and estrogen), glycoprotein (human chorionic gonadotropin; hCG) and protein (human placental lactogen; hPL) hormones were determined by radioimmunoassay of the medium. In rat granulosa and luteal cell cultures, I examined the effect of a number of hormones and second messengers. Effects of follicle-stimulating hormone (FSH), luteinizing hormone (LH), cyclic adenosine monophosphate (cAMP), GnRH and PGF₂[formula omitted] on ovarian hormonogenesis have been previously reported. Changes in cytosolic free calcium concentrations ([Ca²⁺]i) in response to PGF₂[formula omitted] were measured in single rat granulosa and luteal cells. I found that in 34% of granulosa cells, and 53% of luteal cells, there was a 3 to 4 fold increase in resting [Ca²⁺]i within 30 seconds of administration of PGF₂[formula omitted]. Many cells which responded to PGF₂[formula omitted] also responded to GnRH (39% of granulosa cells; 67% of luteal cells). The immediate source of the increased [Ca²⁺]i appeared to be common intracellular stores. No change in hormone production in response to GnRH in placental cell cultures was seen. Trypsin dispersion may have damaged cell surface receptors, therefore the effect of second messengers on hormone production in these cultures was examined. In term and first trimester trophoblast cultures, I observed the following effects with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP): inhibited estrogen production from the supplied androgen precursors; stimulated hCG production; stimulated hPL production in first trimester placental cell cultures (hPL was not measured in enough term cultures to determine the effect of 8-br-cAMP), and stimulated progesterone production. I also investigated the effects of activators and inhibitors of the phosphoinositide (PtdIns(4,5)P₂) breakdown second messenger pathway (TPA, A23187, arachidonic acid); no effects of these agents were seen. Other hormones suspected of having endocrine, paracrine or autocrine effects in the placenta were tested without effect. I conclude that GnRH and PGF₂[formula omitted] cause increases in [Ca²]i in rat ovarian cells, from common intracellular stores of calcium, and that the production of hormones by the human placenta may be under regulation of an agent or agents which induce production of cAMP. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

The dual objectives of this thesis were to study the physiological role of GIP in the enteroinsular axis and to chemically characterize a side fraction identified in the purification of GIP. In the physiological studies, the dependency of the insulinotropic action of GIP on the prevailing state of glycaemia was confirmed in dogs using a system of steady state hyperglycaemia. GIP, from both exogenous and endogenous sources, was demonstrated to potentiate insulin release in the presence of moderate hyperglycaemia. Both glucose and fat administered enterally released immunoreactive-GIP (IR-GIP) and potentiated immunoreactive insulin (IRI) release during moderate hyperglycaemia (150 mg% above basal). Intravenous administration of GIP at 2.0 μg/kq.h was also capable of eliciting insulinotropic action during moderate hyperglycaemia. A mixture of ten amino acids was demonstrated to potentiate insulin release with mild hyperglycaemia (40 mg% above fasting) regardless of routes (intravenous, intraduodenal and oral) of administration. However, the release of IR-GIP was not demonstrated following the administration of the amino acid mixture. Arginine and alanine infused individually did not potentiate insulin release in the presence of mild hyperglycaemia. Intraduodenal hydrochloric acid infusion was also demonstrated not to release IR-GIP in the presence of mild hyperglycaemia. The interactions of GIP with tricarboxylic acid cycle intermediates and pyruvate were studied in euglycaemic conditions in dogs. Intravenous administration of individual metabolites (a-ketoglutaric acid, succinate and pyruvate) on an equimolar basis were shown not to be insulinotropic in the presence or the absence of concurrent GIP infusion (0.4 μg/kg.h). The presence of a minor peptide component in the stage III GIP was initially identified by thin layer chromatography. Confirmation of the presence of this minor component was obtained from polyacrylamide-urea gel. These techniques were unsuitable for preparative separation, as were conventional gel filtration and ion exchange separation. Further purification of GIP on high pressure liquid chromatography indicated the presence of a 5% minor peptide component which was eventually shown to contain two less amino acid residues (tyrosine and alanine) than GIP. The amino acid sequence of GIP III indicated the presence of a peptide component with an amino acid sequence different from GIP in that the first two amino acid residues of the N-terminal portion of the molecule (tyrosine and alanine) were missing. The lack of inhibitory activity to pentagastrin-stimulated acid secretion by synthetic GIP led to a reinvestigation of the amino, acid sequence of the molecule. The work in collaboration with Jornvall (Sweden) indicated an error, in that the original sequence included a second glutamine in position 30. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The interaction of the intestinal insulinotropic hormone GIP (Glucose-dependent Insulinotropic Polypeptide or Gastric Inhibitory Polypeptide) with the stimulus-secretion coupling mechanism of glucose-induced insulin secretion was investigated using the isolated, perfused, rat pancreas technique. The action of GIP in potentiating insulin secretion which had been initiated by a number of metabolic compounds other than glucose (D-glycer-aldehyde, 2-ketoisocaproate, L-leucine + L-glutamine) and the concentration-dependent nature of this action led to the formulation of the hypothesis that the insulinotropic effect of GIP required prior oxidation of these insulin-stimulating metabolites. It was therefore likely that the mechanism whereby GIP potentiated glucose-induced insulin secretion required an interaction located at a level involving effects secondary to glucose degradation in the B-cell. This effect may have required an intact N-terminus on the GIP molecule since a GIP₃-₄₂ homologue, which lacked the N-terminal Tyrosine-Alanine, possessed greatly diminished insulinotropic activity. Additional biological actions of GIP were suggested by the stimulation by the hormone of pancreatic somatostatin release and of the release of lipoprotein lipase-like activity from isolated rat adipocytes. A technique using reversed phase high pressure liquid chromatography (HPLC) was developed allowing the purification and fodination-state analysis of a pure bioactive ¹²⁵I-GIP molecule. Preliminary investigations of receptor binding activity of this purified ¹²⁵I-GIP to isolated rat adipocytes were also performed. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

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The central role of the mammalian heart in the circulation was recognised by Nei Chung when he wrote in his Third Book of the Yellow Emperor's Classic of Internal Medicine (2698 - 2598 B.C.): Treatise on the Five Viscera in Relation their part in Perfecting Life, "The Heart is in accord with the pulse. The complexion of a person shows when the heart is in a splendid condition. The heart rules over the kidneys. . . . . . . . The importance of assessing the functional state of the heart was realised even by the early Egyptian physicians in the 17th Century B.C. as described in the Ebers Papyrus:- "The begining of the physician, secret : knowledge of the heart's movements and knowledge of the heart. There are vessels from it to every limb. As to this, when any physician, any surgeon, or any exorcist applies the hands or his fingers to the head, to the back of the head, to the hands to the place of the stomach, to the arms or to the feet, then he examines the heart, because all his limbs possess its vessels, that is, the heart speaks out of the vessels of every limb".

The existence of a stimulatory intestinal phase of gastric acid secretion has been suspected for some time, and recently the importance of this phase has been recognized. The intestinal phase is of particular interest in relation to the profound gastric hypersecretion associated with portacaval anastomosis. The results of many studies in dogs, and recently in man, have demonstrated conclusively that shunt-related gastric hyper-secretion is due to unmasking of the intestinal phase by hepatic bypass of a humoral stimulant that is normally inactivated by the liver. Definitive experiments have shown that this humoral agent is a hormone that arises in the jejunum. Elaboration of the hormone is triggered both by the entry of food into the jejunum and by a brief period of jejunal distension with a balloon.

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