Two transmembrane signaling mechanisms control expression of the cAMP receptor gene CAR1 during Dictyostelium development

Dictyostelium discoideum is among the best characterized organisms for the study of receptor/guanine nucleotide binding protein-mediated control of differentiation. Dictyostelium grow unicellularly but form fully differentiated multicellular organisms through a developmental program regulated by secreted cAMP activating specific cell-surface receptors. Dictyostelium respond differentially to cAMP at different developmental stages. During early development, expression of certain genes is induced by low-level oscillations of extracellular cAMP. Later, continuous, high cAMP concentrations will promote expression of specific genes in multicellular structures. Here, we show that the cAMP receptor gene CAR1, which is essential for development, utilizes two promoters that are activated at distinct stages of development and respond to different extracellular cAMP conditions. One promoter is active with low-level oscillations of cAMP; exposure to high cAMP concentrations will repress this promoter and induce a second promoter. The CAR1 mRNAs are alternatively spliced but encode identical proteins. Thus, through differential sensitivity to its own ligand, cAMP, two promoters and alternative splicing regulate CAR1 expression during Dictyostelium development.     

Regulation of chorionic gonadotropin gene expression

This paper describes the development of a high-performance liquid chromatographic method for the quantitation of free carnitine, total carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine and octanoylcarnitine in human urine. Carnitine and acylcarnitines were isolated from 10 or 25 microliters of urine using 0.5-ml columns of silica gel, derivatized with 4'-bromophenacyl trifluoromethanesulfonate and separated by high-performance liquid chromatography. Using 4-(N,N-dimethyl-N-ethylammonio)-3-hydroxybutanoate ("e-carnitine") as the internal standard, standard curves (10-300 nmol/ml) were generated. Carnitine and acylcarnitines were quantified (when they were present) in normal human urine and the urine of patients diagnosed with one of three different disorders of organic acid metabolism: methylmalonic aciduria, isovaleric aciduria, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.     

Neuroendocrine mechanisms underlying the control of gonadotropin secretion by steroids

There is considerable evidence that although estradiol may trigger the preovulatory surge of gonadotropins, progesterone is required for its full magnitude and duration and that glucocorticoids bring about selective follicle-stimulating hormone release. The luteinizing hormone-releasing hormone (LHRH) neuron does not have steroid receptors and is regulated by excitatory amino acid neurotransmission. Steroids do not appear to modulate excitatory amino acid receptors directly but increase release of glutamate in the preoptic area. This may be due to the suppression by steroids of the enzyme glutamatic acid decarboxylase67 that converts glutamate into GABA. NMDA receptors colocalize with nitric oxide synthase-containing neurons that surround the LHRH neurons in the preoptic area and intersect the LHRH fibers in the median eminence. Other potential novel pathways of LHRH release that are currently being explored include carbon monoxide generated by the action of heme oxygenase-2 on heme molecules and bradykinin acting via bradykinin B2 receptors.     

Elevated neuropeptide Y gene expression and release during hypoglycemic stress

Our previous studies show that neuropeptide Y (NPY) is involved in mediating sympathetic nerve stimulation-induced vasoconstriction. Insulin hypoglycemia is known to produce increased sympathetic output and elevated arterial pressure. The present study examined the role of NPY in the hypertensive response to insulin by examining the effects of insulin on NPY gene expression, tissue content and release. Subcutaneous injection of insulin produced an immediate increase in plasma NPY immunoreactivity (NPYir) and delayed increases in adrenal and neuronal NPY mRNA and adrenal NPYir in rats. These results suggest that NPY may play a role in insulin-induced hypertension.     

Dihydropyridine receptor gene expression in skeletal muscle from mdx and control mice

The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.     

Homolateral cerebrocortical changes in neuropeptide and receptor expression after minimal cortical infarction

A cortical infarct of 2 mm diameter was obtained in the parietal cortex after a craniotomy, disruption of the dura mater and topical application of 3 M KCl. It has been shown previously that the presence of a small cortical infarct induces an increase in immediate early gene messenger RNA expression followed by an increase in neuropeptide and glutamic acid decarboxylase messenger RNA expression. Glutamate, acting at N-methyl-D-aspartate receptors, is held responsible for these changes, since they are blocked by pretreatment with dizocilpine. In the present study, we have analysed the consequences of the dramatic changes in messenger RNA expression on the level of immediate early gene products c-fos and zif 268, and on that of neuropeptides by using immunohistochemistry. After just 1 h, an increase in c-fos- and zif 268-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct, and is no longer detected after 6 h. An increase in cholecystokinin octapeptide-, substance P-, neuropeptide Y- and somatostatin-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after 30 days. To investigate if these dramatic increases in neuropeptide immunoreactivities may have functional consequences, we studied the level of cholecystokinin receptors by autoradiographic binding using [125I]cholecystokinin-8S and in situ hybridization for the detection of cholecystokinin-b receptor messenger RNA. A decrease in cholecystokinin binding sites and cholecystokinin-b receptor messenger RNA is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after nine days. This study shows that a topical stimulation has diffuse effects, reaching regions far from the site of the lesion, and some of them are still strongly present after nine days. The increase in neuropeptide messenger RNAs is followed by an increase in the protein products of these genes, which may modify the neurotransmission. As a corollary to this, a decrease in cholecystokinin binding sites occurs. This may have further consequences on signal transduction pathways. This decrease in cholecystokinin binding sites is associated with a decrease in the cholecystokinin-b receptor messenger RNA, and this is the first example of a decrease in messenger RNA levels in this experimental model.     

Acute and chronic mechanisms for regulating proximal tubule angiotensin II receptor expression

An NP(X)nY motif is highly conserved among G protein-coupled receptors and is similar to an NPXY motif involved in receptor-mediated endocytosis for several non-G protein-coupled receptors. We investigated the role of this motif in alpha1B-adrenergic receptor function and regulation. Y348A alpha1B-adrenergic receptors in which this sequence was mutated from NPIIY to NPIIA were prepared by site-directed mutagenesis and transfected into Chinese hamster ovary cells. Binding of the antagonist prazosin to Y348A receptors was similar to that of wild-type receptors, but affinity of the Y348A receptors for the agonist epinephrine was increased by approximately 10-fold. Despite this increase in agonist binding affinity, the Y348A mutation completely uncoupled the receptors from stimulation of phosphoinositide hydrolysis and mobilization of intracellular Ca2+. Exposure of cells expressing Y348A receptors to the agonist epinephrine resulted in receptor "sequestration," defined as a loss of cell surface receptors accessible to radioligand in binding assays with intact cells on ice, similar to that for the wild-type receptor. In contrast, Y348A receptors did not undergo "endocytosis" into the light vesicle fraction in sucrose density gradient centrifugation assays, as did the wild-type receptor. These results (i) indicate an important role for Tyr348 in coupling the alpha1B-adrenergic receptor to G protein and subsequent effector activation, (ii) provide further evidence that alpha1B-adrenergic receptor internalization can be separated into a sequestration step and an endocytosis step, (iii) indicate that effector activation and second messenger formation are not required for the sequestration of these receptors but may be involved in endocytosis, and (iv) provide a useful new tool for further investigation of the nature of the subcellular compartments and the molecular modifications involved in the multiple steps involved in internalization of G protein-coupled receptors.     

Recent data on hepatic lipase gene. Structure and functions of this enzyme

Hepatic lipase (HL) is almost exclusively synthesized in the liver. Its gene is located on chromosome 15 in man, on chromosome 9 in rat, and has 9 exons and 8 introns. Inhibitory and activator sequences have been found at the 5' end of the gene, upstream to the initiation site, as well as several consensus sequences such as TRE, SRE, AP-2, CEBP, ERE, AF1 and OCT-1. Glycosylation is an essential step for full active mature protein. The catalytic site is in the N-terminal part of the lipase gene while the lipid binding site is at its C-terminal end. HL is generally thought to be active as a dimer. This enzyme hydrolyses the acyl ester bonds of glycerides and phospholipids as well as the acyl CoA-thiol ester bonds. After being secreted by the hepatocytes, HL remains on the surface of hepatic endothelial cells and hepatocytes, bound to heparan sulfate proteoglycans, where it acts on the uptake of chylomicron remnants, IDL and HDL cholesterol ester. HL also participates in the VLDL to IDL and LDL cascade and in the conversion of HDL2 to HDL3 and pre-beta 1 HDL. Thus, HL plays an important role in lipoprotein metabolism and in reverse cholesterol transport and may thus be involved in atherogenic processes. Moreover, HL expression is regulated by hormones and nutritional state in the pre- and post-natal periods. Therefore, it appears of interest to gain further insight into the regulation of HL gene expression in order to better understand plasma lipoprotein metabolism.     

Regulation of gene expression by corticoid hormones in the brain and spinal cord

Glucocorticoids (GC) and mineralocorticoids (MC) have profound regulatory effects upon the central nervous system (CNS). Hormonal regulation affects several molecules essential to CNS function. First, evidences are presented that mRNA expression of the alpha3 and beta1-subunits of the Na,K-ATPase are increased by GC and physiological doses of MC in a region-dependent manner. Instead, high MC doses reduce the beta1 isoform and enzyme activity in amygdaloid and hypothalamic nuclei, an effect which may be related to MC control of salt appetite. The alpha3-subunit mRNA of the Na,K-ATPase is also stimulated by GC in motoneurons of the injured spinal cord, suggesting a role for the enzyme in GC neuroprotection. Second, we provide evidences for hormonal effects on the expression of mRNA for the neuropeptide arginine vasopressin (AVP). Our data show that GC inhibition of AVP mRNA levels in the paraventricular nucleus is sex-hormone dependent. This sexual dimorphism may explain sex differences in the hypothalamic-pituitary-adrenal axis function between female and male rats. Third, steroid effects on the astrocyte marker glial fibrillary acidic protein (GFAP) points to a complex regulatory mechanism. In an animal model of neurodegeneration (the Wobbler mouse) showing pronounced astrogliosis of the spinal cord, in vivo GC treatment down-regulated GFAP immunoreactivity, whereas the membrane-active steroid antioxidant U-74389F up-regulated this protein. It is likely that variations in GFAP protein expression affect spinal cord neurodegeneration in Wobbler mice. Fourth, an interaction between neurotrophins and GC is shown in the injured rat spinal cord. In this model, intensive GC treatment increases immunoreactive low affinity nerve growth factor (NGF) receptor in motoneuron processes. Because GC also increases immunoreactive NGF, this mechanism would support trophism and regeneration in damaged tissues. In conclusion, evidences show that some molecules regulated by adrenal steroids in neurons and glial cells are not only involved in physiological control, but additionally, may play important roles in neuropathology.     

Distribution of a novel hypothalamic neuropeptide Y receptor gene and it''s absence in rat

The alteration in calcium transport in the liver of rats with streptozocin(STZ)-diabetic state was investigated. STZ (6 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 2 weeks later they were sacrificed by bleeding. STZ administration caused a remarkable elevation of serum glucose concentration. Liver calcium content was significantly increased by STZ administration. Hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity was markedly elevated by STZ administration. This increase was completely abolished by the presence of staurosporine (10(-7)-10(-5) M), an inhibitor of protein kinase C, in the enzyme reaction mixture, suggesting an involvement of protein kinase C signalling. Moreover, the STZ-induced increase in liver plasma membrane (Ca2+-Mg2+)-ATPase activity was significantly raised by the presence of okadaic acid (10(-5) and 10(-4) M). Meanwhile, the STZ-increased (Ca2+-Mg2+)-ATPase activity was not appreciably altered by the presence of anti-regucalcin IgG in the reaction mixture, indicating that the activatory protein regucalcin does not participate in the elevation of the enzyme activity. The present study demonstrates that STZ-induced diabetes causes the increase in hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity of rats.     

Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression

In a previous study we have shown that an intravenous infusion of pramlintide (an analogue of human amylin) delayed gastric emptying, but the dose of pramlintide was supraphysiological in relation to the amylin response to food in non-diabetic subjects. The purpose of this study was to examine the dose response relationship of subcutaneous injections of pramlintide on gastric emptying and to determine whether administration of the drug before one meal has an impact on the subsequent meal. Eleven men with insulin-dependent diabetes mellitus were studied in a double-blind, randomised, four-way crossover design. None had autonomic neuropathy. Euglycaemia was maintained overnight before the study day. At -30 min the patients self-injected their usual morning insulin and at -15 min they injected the study drug (either placebo or 30, 60 or 90 microg pramlintide) subcutaneously. At 0 min they ate a standard meal consisting of a pancake, labelled with 99mTc, and a milkshake containing 3-ortho-methylglucose (3-OMG). Gastric emptying images were obtained for the next 8 h. At 240 min the subjects ate a similar meal, but on this occasion the pancake was labelled with (111)In. All three doses of pramlintide delayed emptying of the solid component of the first meal (p < 0.004) with no significant difference between the drug doses. There were no differences between placebo and pramlintide after the second meal. All three doses of pramlintide resulted in a prolongation in the time to peak plasma 3-OMG level (p < 0.0001) after the first meal but there was no difference after the second meal.     

Progesterone receptor gene expression in craniopharyngiomas and evidence for biological activity

OBJECTIVE: Previous studies have demonstrated the presence of estrogen receptors in human craniopharyngiomas, raising the possibility that these lesions can be influenced by steroids. To complement these earlier findings, we examined for the presence of progesterone receptor (PR) messenger RNA in surgically removed craniopharyngiomas and performed some studies to determine whether progestogens can exert biological effects on these tumors in vitro. METHODS: Total RNA was extracted from fresh surgically removed craniopharyngiomas and reverse-transcribed into cDNA. The polymerase chain reaction was applied to this craniopharyngioma-derived cDNA using amplimers complementary to exons 4 and 7 of the PR gene. Additionally, craniopharyngioma cell cultures were established, and the in vitro effects of progesterone and 6 alpha-methyl-17 alpha-hydroxyprogesterone acetate on [3H]thymidine uptake and 17 beta-estradiol oxidoreductase activity were determined. RESULTS: Reversed-transcribed polymerase chain reaction of craniopharyngioma-derived RNA yielded bands of predicted size (389 base pairs) in six of seven tumors studied. Hinfl digestion and direct sequencing of the bands confirmed that the polymerase chain reaction DNA was representative of PR messenger RNA. Treatment of craniopharyngioma cell cultures with progesterone resulted in reduced [3H]thymidine uptake. Both progesterone and 6 alpha-methyl-17 alpha-hydroxyprogesterone acetate powerfully increased oxidative 17 beta-estradiol oxidoreductase activity. CONCLUSION: These results provide evidence that PR messenger RNA can be produced by at least some human craniopharyngiomas and indirectly show that this is translated into biologically active receptor protein.     

Elevated corticosterone is not required for the rapid induction of neuropeptide Y gene expression by an overnight fast

Fasting stimulates corticosterone (B) secretion and the expression and secretion of hypothalamic neuropeptide Y in rats. These studies tested the hypothesis that the rapid and marked fasting-induced increases in plasma B are responsible for stimulation of neuropeptide Y (NPY) gene expression. Plasma leptin and insulin were measured because they are also signals known to affect NPY messenger RNA (mRNA). Intact or adrenalectomized rats given a low fixed level of corticosterone (B replaced) were fasted for 48 h. NPY mRNA in the mediobasal hypothalamus, measured by nuclease protection assay, was elevated similarly above ad lib-fed controls in both intact and B replaced groups at 15 and 48 h after the onset of fasting. NPY immunoreactivity in the mediobasal hypothalamus increased between 3 and 48 h after onset of the fast in intact but not in B replaced groups. The fasting-induced decreases in leptin observed in intact rats at 48 h did not occur in B replaced rats. Fasting-induced decreases in insulin occurred in B replaced rats but not in intact rats. We conclude that: 1) elevated B is not required for fasting-induced increases in hypothalamic NPY gene expression; and 2) decreases in neither leptin nor insulin alone signal the changes that occur in NPY mRNA in fasted rats.