Peter A. Janssens

The Gene for a Novel Member of the Whey Acidic Protein Family Encodes Three Four-disulfide Core Domains and Is Asynchronously Expressed during Lactation

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of thetWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.

Gonadal hormones inhibit the induction of metamorphosis by thyroid hormones in Xenopus laevis tadpoles in vivo, but not in vitro

Although the major hormones controlling amphibian metamorphosis are those of the thyroid, other hormones, notably prolactin and the adrenal steroids, modulate the effects of thyroid hormones (TH). Some authors report that the gonadal steroids stimulate the metamorphic actions of TH whereas others report inhibition. The aims of the present study were to determine the effects of gonadal steroids on TH-induced metamorphosis in Xenopus laevis and to determine the site of action of these steroids. In all cases, hormones were added to the water in which the tadpoles were swimming. The gonadal steroids, testosterone and 17 beta-estradiol, inhibited triiodothyronine (T3)-induced metamorphosis in living, premetamorphic tadpoles of X. laevis. Both steroids, at 3.4 microM, prevented the reduction in body weight and the shrinkage of head and alimentary canal brought about by 1 nM T3. In contrast, 3.4 microM corticosterone stimulated T3-induced metamorphosis. Addition of 100 nM T3 to the medium induced a large reduction in size of X. laevis tails cultured in vitro. The antagonistic effects of testosterone were not reproduced in such cultures, whereas the synergistic action of corticosterone was maintained. Testosterone had no effect upon the specific binding of T3 to X. laevis tail tissue, whereas corticosterone increased such binding. These findings indicate that, while corticosterone stimulates the metamorphic actions of T3 by acting directly in the peripheral tissues, the gonadal steroids, particularly testosterone, inhibit T3 by acting at a more central site. Prolactin is known to antagonize the metamorphic actions of T3 and one such central action could be the stimulation of prolactin synthesis. However, testosterone inhibited the prometamorphic actions of bromocriptine, which stimulates metamorphosis by inhibiting production of prolactin. Thus the central action of testosterone is unlikely to be a stimulation of prolactin production.

Hormonal control of glycogenolysis and the mechanism of action of adrenaline in amphibian liver in vitro

In in vitro cultures of liver from Ambystoma mexicanum glycogenolysis was stimulated by adrenaline, glucagon, and vasopressin in a dose-dependent manner. Maximum activity was seen at 10(-6) M hormone while 10(-9) M was without effect. Dibutyryl cyclic AMP (10(-3) M) stimulated glycogenolysis maximally although 10(-5) M had no effect. The glucose release brought about by adrenaline was blocked by the beta-adrenergic antagonist propranolol but not by prazosin or yohimbine which are alpha 1- and alpha 2-adrenergic antagonists. Cyclic AMP concentrations in liver were elevated within 1 min of administration of adrenaline and remained elevated for at least 60 min. Phosphorylase a activity was elevated 10 min after addition of adrenaline and remained elevated for at least 6 hr. The rise in hepatic cyclic AMP concentration and phosphorylase a activity was largely blocked by propranolol. These findings are consistent with adrenaline acting via a beta-adrenergic receptor in A. mexicanum. Glycogenolysis in A. mexicanum liver was stimulated by isoprenaline and phenylephrine and in each case the stimulation was reduced in the presence of propranolol but unaffected by phentolamine. High concentrations of methoxamine, a specific alpha 1-agonist, had no effect upon glycogenolysis. These findings suggest that alpha-adrenergic receptors play no role in regulation of glycogenolysis in A. mexicanum.

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.

Binding of adrenergic ligands to liver plasma membrane preparations from the axolotl, Ambystoma mexicanum; the toad, Xenopus laevis; and the Australian lungfish, Neoceratodus forsteri

The beta-adrenergic ligand iodocyanopindolol (ICP) bound specifically to hepatic plasma membrane preparations from the axolotl, Ambystoma mexicanum (Bmax, 40 fmol/mg protein (P) at free concentration above 140 pM; KD, 42 pM); the toad, Xenopus laevis (Bmax, 200 fmol/mg P at 1 nM; KD, 300 pM); and the Australian lungfish, Neoceratodus forsteri (Bmax, 100 fmol/mg P at 5 nM). For the lungfish, the Scatchard plot was curved showing two classes of binding site with KDs of 20 and 500 pM. Neither the alpha 1-adrenergic ligand prazosin nor the alpha 2-adrenergic ligand yohimbine bound specifically to hepatic membrane preparations from any of the three species. Several adrenergic ligands displaced ICP from hepatic membrane preparations of all three species with KDs of Axolotl--propranolol, 50 nM; isoprenaline, 600 nM; adrenaline, 10 microM; phenylephrine, 20 microM; noradrenaline, 40 microM; and phentolamine, greater than 100 microM; X. laevis--propranolol, 30 nM; isoprenaline, 100 microM; adrenaline, 200 microM; noradrenaline, 300 microM; phenylephrine, 1 mM; and phentolamine, greater than 1 mM; N. forsteri,--propranolol, 25 nM; isoprenaline, 1 microM; adrenaline, 20 microM; phenylephrine, 35 microM; noradrenaline, 600 microM; and phentolamine, 400 microM. These findings suggest that alpha-adrenergic receptors are not present in hepatic plasma membrane preparations from these three species and that the hepatic actions of catecholamines are mediated via beta-adrenergic receptors. The order of binding of the beta-adrenergic ligands suggests that the receptors are of the beta 2 type.

Effects of fasting and cortisol administration on carbohydrate metabolism in Xenopus laevis Daudin

The concentrations of most intermediates of glycolysis and gluconeogenesis were measured in livers from juvenile and adult Xenopus laevis. Comparison of the mass action ratios of the enzymes of these pathways with their apparent equilibrium constants suggests that flow through the pathways is most likely to be regulated at the hexokinase/glucose-6-phosphatase (G-6-Pase) or the pyruvate kinase/pyruvate carboxylase + phosphoenolpyruvate carboxykinase (PEPCK) steps. Injection of 10 mg of cortisol/kg daily for 7 days or fasting for up to 130 days had no effect upon the mass action ratio of any enzyme. In juvenile X. laevis fasted for 50 days, liver weight, blood glucose, and liver glycogen all decreased. Fructose-1,6-diphosphatase (FDPase) activity increased slightly while G-6-Pase activity decreased. Injection of cortisol (25 mg/kg daily for 7 days) was followed by increases in liver glycogen and blood glucose and in the rate of urea excretion. G-6-Pase activity rose above that in fed toads. These results are consistent with cortisol causing an increase in glucose synthesis, regulated at G-6-Pase. In adult X. laevis fasted for 60 days, hepatic alanine aminotransferase (AAT) activity fell by half but there were no changes in the activity of other enzymes or in the levels of glycolytic intermediates. After 130 days fasting, plasma free fatty acid and urea levels were reduced but there were no changes in plasma glucose or amino acid concentration or in liver or muscle glycogen content. The activities of G-6-Pase, FDPase, AAT, and carbamyl phosphate synthetase were reduced but glutamate dehydrogenase and total PEPCK were unchanged. In fed adult toads, injection of cortisol (10 mg/kg daily for 7 days) had no effect on any of the parameters measured while in toads fasted for 60 days, the only changes following injection of cortisol were increases in the concentration of urea in the plasma and in its rate of excretion, together with an increase in the activity of hepatic PEPCK. The results suggest that the effects of fasting and cortisol injection vary with the age of the animals. Considerable variability in the results, probably attributable to the low metabolic rate of the toads and seasonal effects, may well be masking the effects of administration of cortisol.

The role of hormones in regulation of carbohydrate metabolism in the Australian lungfish Neoceratodus forsteri

Adrenaline, arginine vasopressin, arginine vasotocin, and glucagon, all at 10(-6) M, stimulate glycogen breakdown and glucose release from hepatic tissue of Neoceratodus forsteri cultured in vitro. Adrenaline acts via a beta-adrenergic receptor in this species. Gluconeogenesis from 2 mM lactate occurred at a rate of 680 +/- 39 nmol/g liver/h. This rate of gluconeogenesis was unaffected by the addition of 10(-6) M adrenaline.

Hormonal regulation of hepatic glycogenolysis in the toad, Xenopus laevis, is mediated by cyclic AMP and not Ca2+

Hepatic glycogenolysis and glycogen phosphorylase a activity were stimulated by arginine vasotocin (AVT) in liver pieces from Xenopus laevis cultured in vitro. In each case, the EC50 was about l nM. The increased rate of glycogenolysis brought about by either AVT or adrenaline was maintained for at least 6 hr and was unchanged when Ca2+ salts were omitted from the medium or when 2.5 mM EGTA was added. Neither the Ca2+ ionophore, A23187, nor the Ca2+ channel blocker, verapamil, had any effect on the rate of glycogenolysis in the presence or the absence of either hormone. Tissue cyclic AMP levels were unchanged by addition of AVT alone but were doubled in the presence of AVT plus either of the phosphodiesterase inhibitors, isobutylmethylxanthine or RO20-1724. These findings suggest that hormones regulating hepatic glycogenolysis in X. laevis use cyclic AMP, and not Ca2+, as an intracellular messenger. We would argue that cytosolic Ca2+ may not have become involved in regulation of hepatic glycogenolysis until after the ancestors of present day amphibians separated from those of present day mammals.

Lymphocytes and MHC class II positive cells in the female rabbit reproductive tract before and after ovulation

In this study, we identified lymphocytes and MHC class II positive (MHC‐II+) cells in the reproductive tract of female rabbits both before and after ovulation. CD43+ T cells were frequently present in the mucosa of the oviduct, cervix, and vagina, but far fewer positive cells were seen in the endometrium. The induction of ovulation did not change the cell density in these regions. KEN‐5+ T cells and MHC‐II+ cells were also frequently seen in the mucosa of the oviduct, cervix, and vagina both before and after ovulation. However, in the uterus, there were very few positive cells before ovulation, but the number increased dramatically after ovulation. Associated with the increase of KEN‐5+ T cells, IL‐2 mRNA expression in the uterus also increased after ovulation, suggesting that the uterus experienced an increase of T‐cell activation. IgM‐ and IgA‐positive B cells were not commonly seen in the reproductive tract and the induction of ovulation did not alter this. Our results suggest that the reproductive tract of female rabbits has the capacity to mount an immune response and that the immune cell distribution of the rabbit reproductive tract has some distinctive features compared with that found in other species.

Glucagon and insulin regulate in Vitro hepatic glycogenolysis in the axolotl Ambystoma mexicanum via changes in tissue cyclic AMP concentration

Glucagon increases the rate of glycogenolysis in in vitro cultures of hepatic tissue from the axolotl Ambystoma mexicanum. The hormone causes an increase in the concentration of cyclic AMP in the tissue which is followed by activation of glycogen phosphorylase and subsequent breakdown of glycogen and release of glucose from the tissue. Insulin counteracts the glycogenolytic effect of glucagon by inhibiting the increase in tissue cyclic AMP concentration brought about by glucagon. This inhibitory effect of insulin is not seen in the presence of the phosphodiesterase inhibitor IBMX and so it appears that the initial action of insulin is a stimulation of cyclic AMP phosphodiesterase activity which lowers the tissue concentration of cyclic AMP and so counters the actions of hormones that act by raising the tissue concentration of cyclic AMP. This model for the mode of action of insulin is supported by the finding that insulin also interferes with the glycogenolytic actions of adrenaline, a second hormone which acts by raising tissue cyclic AMP concentrations.