Molecular analysis of the koala reproductive hormones and their receptors: gonadotrophin-releasing hormone (GnRH), follicle-stimulating hormone ß and luteinising hormone ß with localisation of GnRH.

During evolution, reproductive hormones and their receptors in the brain-pituitary-gonadal axis have been altered by genetic mechanisms. To understand how the neuroendocrine control of reproduction evolved in mammals, it is important to examine marsupials, the closest group to placental mammals. We hypothesised that at least some of the hormones and receptors found in placental mammals would be present in koala, a marsupial. We examined the expression of koala mRNA for the reproductive molecules. Koala cDNAs were cloned from brain for gonadotrophin-releasing hormones (GnRH1 and GnRH2) or from pituitary for GnRH receptors, types I and II, follicle-stimulating hormone (FSH)ß and luteinising hormone (LH)ß, and from gonads for FSH and LH receptors. Deduced proteins were compared by sequence alignment and phylogenetic analysis with those of other vertebrates. In conclusion, the koala expressed mRNA for these eight putative reproductive molecules, whereas at least one of these molecules is missing in some species in the amniote lineage, including humans. In addition, GnRH1 and 2 are shown by immunohistochemistry to be expressed as proteins in the brain.

Cell volume affects glycogen phosphorylase activity in fish hepatocytes.

The activity of glycogen phosphorylase (GPase) in the active a-form (GPase a) is dependent on the hydration state of hepatocytes. We establish that GPase a catalysis in catfish (Ameiurus nebulosus) hepatocytes is a function of medium osmolarity and that a linear relationship exists between GPase a activity and osmolarity between 254 mosmol l(-1) and 478 mosmol l(-1). Exposure of isolated hepatocytes to hyperosmotic media increases enzyme activity up to 7-fold, indicative of covalent phosphorylation. GPase activation associated with cell shrinkage peaks within 10 min of exposure. The average degree of activation (2.7-fold-increase of GPase a) is only slightly less than in hepatocytes exposed to glucagon (3.1-fold-increase) under isosmotic conditions; with glucagon, the maximum is reached within 2 min. Phosphorylation status remains elevated during the entire 40 min experimental period; cells do not undergo regulatory volume increase (RVI) during this period and do not regain pre-exposure volume. We interpret the increased GPase a activity as an inherent response to hyperosmotic stress, likely brought about by molecular crowding. Activation of the enzyme results in increased glucose production from endogenous glycogen. Glucose is not retained in the liver cells, but may act as an oxidative substrate in extrahepatic tissues for the increased metabolic demand of ion regulation. Protein kinase A or intracellular Ca(2+) make apparently small contributions to the activation of GPase, leaving us to speculate on alternate routes of enzyme activation. Conversely, hepatocyte swelling in hyposmotic medium leads to significant decreases in GPase a activity and curtailed glucose output. A minimum is attained in 10 min, and pre-insult rates are re-established within 40 min, somewhat lagging behind readjustment in cell volume by regulatory volume decrease (RVD). We conclude that cell swelling and subsequent RVD do not signify stress to the cells and metabolic demand may be decreased under cell swelling conditions. Alteration of GPase phosphorylation with extracellular osmolarity appears to be a general phenomenon, since we also find it in hepatocytes of another freshwater catfish (Clarias batrachus) and a marine scorpaenid (Sebastes caurinus).

Glutamine synthetase in tilapia gastrointestinal tract: zonation, cDNA and induction by cortisol.

Glutamine synthetase, an enzyme generally associated with ammonia detoxication in the vertebrate brain and with hepatic nitrogen turnover in mammals, shows substantial activities in the gastrointestinal tract of teleostean fishes. Enzyme activity is highest in the central area of the stomach and reveals a distinct distribution pattern in stomach and along the intestine of tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and copper rockfish (Sebastes caurinus). In all three species, intestinal activity peaks in the distal region of the intestine. The brain contains the highest titre of the enzyme (46 U g(-1) in tilapia brain versus 15 U g(-1) in tilapia stomach), but because of the relative mass of the stomach, the largest glutamine synthetase pool in tilapia body appears to be localized in the stomach. Activities in white and red muscle are very modest at 0.1% of the brain. Independent of distribution, peak activities of glutamine synthetase in selected areas of tilapia stomach and intestine are significantly (two- to fourfold) increased after a 5-day treatment with an intraperitoneal cortisol deposit. Cortisol also increases glutamine synthetase activity in tilapia liver, white and red muscle, while activities in brain remain unaffected. We cloned and sequenced the predominant transcript of tilapia stomach glutamine synthetase (about 1.9 kb), encoding a 371-amino acid peptide. The open reading frame shows considerable identity with glutamine synthetase in toadfish (92% at peptide level, 87% at nucleotide level), but possesses a longer 3'-untranslated region than the toadfish. The tilapia glutamine synthetase mRNA contains a remnant of a putative mitochondrial leader sequence, but without a conserved second site for initiation of translation. We also find evidence for additional transcripts of glutamine synthetase in tilapia, suggesting multiple genes. Finally, we present evidence for similar abundance of glutamine synthetase transcripts in all regions of rockfish intestine. The physiological significance of the presence of glutamine synthetase in teleostean intestine is discussed.

Novel role for prostaglandin E2 in fish hepatocytes: regulation of glucose metabolism.

Prostaglandin E(2) (PGE(2)) potently activated glycogenolysis and gluconeogenesis in isolated rockfish (Sebastes caurinus) hepatocytes. The average degree of activation for glycogenolysis was 6.4+/-0.67-fold (mean+/-S.E.M.; n=37), and could be as much as 19-fold. Analysis of dose-concentration relationships between glycogenolytic actions and PGE(2) concentrations yielded an EC(50) around 120 nM in hepatocyte suspensions and 2 nM for hepatocytes immobilized on perifusion columns. For the activation of gluconeogenesis (1.74+/-0.14-fold; n=10), the EC(50) for suspensions was 60 nM. Intracellular targets for PGE(2) actions are adenylyl cyclase, protein kinase A and glycogen phosphorylase. Concentrations of cAMP increased with increasing concentrations of PGE(2), and peaked within 2 min of hormone application. In the presence of the phosphodiesterase inhibitor, isobutyl-3-methylxanthine, peak height was increased and peak duration extended. The protein kinase A inhibitor, Rp-cAMPS, counteracted the activation of glycogenolysis by PGE(2), implying that the adenylyl cyclase/protein kinase A pathway is the most important, if not exclusive, route of message transduction. PGE(2) activated plasma membrane adenylyl cyclase and hepatocyte glycogen phosphorylase in a dose-dependent manner. The effects were specific for PGE(2); smaller degrees of activation of glycogenolysis were noted for PGE(1), 11-deoxy PGE(1), 19-R-hydroxy-PGE(2), and prostaglandins of the A, B and Falpha-series. The selective EP(2)-receptor agonist, butaprost, was as effective as PGE(2), suggesting that rockfish liver contains prostaglandin receptors pharmacologically related to the EP(2) receptors of non-hepatic tissues of mammals. Rockfish hepatocytes quickly degraded added PGE(2) (t((1/2))=17-26 min). A similar ability to degrade PGE(2) has been noted in catfish (Ameiurus nebulosus) hepatocytes, but no glycogenolytic or gluconeogenic actions of the hormone are noted for this species. We conclude that PGE(2) is an important metabolic hormone in fish liver, with cAMP-mediated actions on glycogen and glucose metabolism, and probably other pathways regulated by cAMP and protein kinase A. The constant presence of EP(2)-like receptors is a unique feature of the fish liver, with interesting implications for function and evolution of prostaglandin receptors in vertebrates.

Improved results in combination chemotherapy of head and neck cancer using a kinetically-based approach: a randomised study with and without adriamycin.

One hundred and seventeen patients with advanced squamous cell carcinoma of the head and neck were randomised between two combination schedules, one with and the other without adramycin. Responses (more than 50% tumor regression) were 67% overall with 63% responding to the combination without adriamycin and 82% responding to the schedule containing it. The increase in response rate seen with the addition of adriamycin is not statistically significant. Prior radiotherapy reduced the likelihood of response to chemotherapy.