Differential ligand binding and agonist-induced regulation characteristics of the two rainbow trout GH receptors, Ghr1 and Ghr2, in transfected cells.

Previously, we isolated and characterized two distinct GH receptor (GHR)-encoding mRNAs, ghr1 and ghr2, from rainbow trout. In this study, Chinese hamster ovary-K1 cells were individually transfected with plasmids that contained cDNAs encoding rainbow trout ghr1 or ghr2. High affinity binding of (125)I-salmonid GH (sGH) by the expressed receptors was saturable, displaceable, and ligand selective. Whole-cell binding analysis revealed a single class of binding site; for Ghr1 K(d)=8 nM, for Ghr2 K(d)=17 nM. While salmonid prolactin (sPrl) displaced (125)I-sGH from both Ghr1 and Ghr2, the affinity of either receptor subtype for sPrl was substantially less than for sGH; salmonid somatolactin, another member of the GH-PRL family, did not displace labeled sGH except at pharmacological concentrations. (125)I-sGH was internalized by Ghr1- and Ghr2-expressing cells in a time-dependent manner; the maximum internalization reached was 71% for Ghr1 and 55% for Ghr2. Long-term exposure (24 h) of transfected cells to sGH up-regulated surface expression of both Ghr1 and Ghr2; however, sGH induced surface expression of Ghr1 to a greater extent than that of Ghr2. These results indicate that rainbow trout ghrs display both overlapping and distinct characteristics that may be important for ligand selection and differential action in target organs.

Dual role of beta-carotene in combination with cigarette smoke aqueous extract on the formation of mutagenic lipid peroxidation products in lung membranes: dependence on pO2.

Results from some intervention trials indicated that supplemental beta-carotene enhanced lung cancer incidence and mortality in chronic smokers. The aim of this study was to verify the hypothesis that high concentrations of the carotenoid, under the pO2 present in lung (100-150 mmHg), may exert deleterious effects through a prooxidant mechanism. To test this hypothesis, we examined the interactions of beta-carotene and cigarette smoke condensate (tar) on the formation of lipid peroxidation products in rat lung microsomal membranes enriched in vitro with varying beta-carotene concentrations (from 1 to 10 nmol/mg prot) and then incubated with tar (6-25 microg/ml) under different pO2. As markers of lipid peroxidation, we evaluated the levels of conjugated dienes and malondialdehyde, possessing mutagenic and pro-carcinogenic activity. The exposure of microsomal membranes to tar induced a dose-dependent enhancement of lipid peroxidation, which progressively increased as a function of pO2. Under a low pO2 (15 mmHg), beta-carotene acted clearly as an antioxidant, inhibiting tar-induced lipid peroxidation. However, the carotenoid progressively lost its antioxidant efficiency by increasing pO2 (50-100 mmHg) and acted as a prooxidant at pO2 ranging from 100 to 760 mmHg in a dose-dependent manner. Consistent with this finding, the addition of alpha-tocopherol (25 microM) prevented the prooxidant effects of the carotenoid. beta-Carotene auto-oxidation, measured as formation of 5,6-epoxy-beta,beta-carotene, was faster at high than at low pO2 and the carotenoid was more rapidly consumed in the presence of tar. These data point out that the carotenoid may enhance cigarette smoke-induced oxidative stress and exert potential deleterious effects at the pO2 normally present in lung tissue.

Ablation of the chemokine monocyte chemoattractant protein-1 delays retrograde neuronal degeneration, attenuates microglial activation, and alters expression of cell death molecules.

The mechanisms regulating retrograde neuronal degeneration and subsequent death of thalamic neurons following cortical injury are not well understood. However, the delay in the onset of retrograde cell death and observed morphological changes are consistent with apoptosis. Our previous studies demonstrated that monocyte chemoattractant protein-1 (MCP-1), a beta-chemokine that attracts cells of monocytic origin to sites of injury, is rapidly and specifically expressed in the lateral geniculate nucleus following visual cortical lesions. To determine the potential role of MCP-1 in retrograde degeneration, the present study examined the effect of genetic deletion of MCP-1 (MCP-1 KO or -/-) or its high affinity receptor CCR2 (CCR2 KO or -/-) on thalamic microglial activation and neuronal cell death following aspiration lesions of the visual cortex in adult mice. Deletion of the MCP-1 gene delayed microglial activation and transiently improved the survival of thalamic neurons. Deletion of the CCR2 receptor resulted in a significant increase in apoptosis as measured by nucleosomal fragmentation after injury compared to wild-type mice, but did not alter neuron survival, suggesting that glial apoptosis is increased in the receptor knockout mice. Investigation of Bcl-2, Bax, Fas, Fas ligand (FasL) and activated caspase-3, key regulators of apoptosis that can be modulated by cytokines, revealed complex alterations of mRNA and protein levels in MCP-1(-/-) and CCR2(-/-) mice. As examples, Bcl-2 protein was detected in wild-type, but not in MCP-1(-/-) mice. Caspase-3 activity was higher in MCP-1(-/-) mice compared to wild-type and CCR2(-/-) mice at 5 days after injury. High levels of activated caspase-3 correlate with the beginning of a period of delayed, but rapid cell death in the thalami of MCP-1(-/-) mice. In summary, our data strongly suggest that MCP-1 is involved in early microglial response to axotomy and that modulation of this chemokine could provide a novel strategy for improved neuronal survival following injury to the central nervous system.

Molecular cloning of cDNA encoding a drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel.

Ryanodine is a plant alkaloid that was originally used as an insecticide. To study the function and regulation of the ryanodine receptor (RyR) from insect cells, we have cloned the entire cDNA sequence of RyR from the fruit fly Drosophila melanogaster. The primary sequence of the Drosophila RyR contains 5134 amino acids, which shares approximately 45% identity with RyRs from mammalian cells, with a large cytoplasmic domain at the amino-terminal end and a small transmembrane domain at the carboxyl-terminal end. To characterize the Ca(2+) release channel activity of the cloned Drosophila RyR, we expressed both full-length and a deletion mutant of Drosophila RyR lacking amino acids 277-3650 (Drosophila RyR-C) in Chinese hamster ovary cells. For subcellular localization of the expressed Drosophila RyR and Drosophila RyR-C proteins, green fluorescent protein (GFP)-Drosophila RyR and GFP-Drosophila RyR-C fusion constructs were generated. Confocal microscopic imaging identified GFP-Drosophila RyR and GFP-Drosophila RyR-C on the endoplasmic reticulum membranes of transfected cells. Upon reconstitution into the lipid bilayer membrane, Drosophila RyR-C formed a large conductance cation-selective channel, which was sensitive to modulation by ryanodine. Opening of the Drosophila RyR-C channel required the presence of microM concentration of Ca(2+) in the cytosolic solution, but the channel was insensitive to inhibition by Ca(2+) at concentrations as high as 20 mM. Our data are consistent with our previous observation with the mammalian RyR that the conduction pore of the calcium release channel resides within the carboxyl-terminal end of the protein and further demonstrate that structural and functional features are essentially shared by mammalian and insect RyRs.

Improvement of cytochrome P450c17 catalytic processivity as a novel mechanism to attenuate hormonal consequences of enzyme decay in the testes after a gonadotropin surge.

OBJECTIVE: The aim of this study was to gain understanding of the apparent discrepancy between the moderate restriction of testosterone synthesizing capacity and the nearly complete decay of the androgen-producing enzyme, cytochrome P450c17 (CYP17; steroid 17 alpha-hydroxylase/17,20-lyase), in rat testes during the desensitization phase induced by a single, high-dose gonadotropin (human chorionic gonadotropin, hCG) injection. DESIGN AND METHODS: Adult male rats received 25 IU hCG i.v., and purified Leydig cells and crude interstitial cell microsomes were prepared 0, 4, 12, 24, 48, 72, 120 and 192 h afterwards. Component CYP17 activities, i.e. simultaneously catalyzed productive androgen formation and abortive 17 alpha-hydroxyprogesterone release, and their ratio (processivity), were compared with CYP17 levels and testosterone secretion rates. RESULTS: Leydig cells isolated 48 h after the artificial hCG surge produce 62% less testosterone than control cells upon stimulation in vitro, though CYP17 levels are reduced by 97%. Its total activity decreases by 87%, resulting in a 4.5-fold rise in the turnover number; the processivity is additionally improved 5-fold over controls. Parallel changes occur in interstitial cell microsomes; a negative linear correlation exists between the ratio of productive over total CYP17 activities and the actual CYP17 concentrations. CYP17 is partly denatured to P420 during hCG action, but other heme proteins (cytochrome b5) remain unchanged. Animal treatment with estradiol results in CYP17 down-regulation without any concomitant effect on enzyme processivity. CONCLUSION: Improved CYP17 processivity is suggested to be the consequence of (otherwise rate-limiting) improved electron transfer efficiency towards CYP17. It explains the relatively high testosterone secretion during Leydig cell desensitization and is interpreted to be a protective mechanism to confine adverse consequences of enzyme decay.

Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein.

The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes a linear conductance chloride channel, which is regulated by cAMP-dependent protein kinase phosphorylation at multiple sites located in the intracellular regulatory (R) domain. Studies in a lipid bilayer system, reported here, provide evidence for the control of CFTR chloride channel by its R domain. The exogenous R domain protein (encoded by exon 13 plus 85 base pairs of exon 14) interacted specifically with the CFTR molecule and inhibited the chloride conductance in a phosphorylation-dependent manner. Only the unphosphorylated R domain protein blocked the CFTR channel. Such functional interaction suggests that the putative gating particle of the CFTR chloride channel resides in the R domain.

The role of vitamin E in the susceptibility of rat lung and liver microsomes to iron-stimulated peroxidation.

The production of thiobarbituric acid-reactive substances (TBA-RS) and ethane, two markers of the lipid peroxidation process, was evaluated in rat lung and liver microsomal membranes incubated in the presence of either ferrous ions or a mixture of ferric ions and ascorbate. Microsomal fractions isolated from lung tissue were more resistant than those isolated from the liver. Compared to Fe2+, the association of Fe3+/ascorbate seemed to be totally ineffective in stimulating peroxidation of lung microsomes. The fatty acid profile of lung and liver microsomal membranes could not be responsible for their different susceptibility to free radical degradation. The microsomal fraction isolated from lung showed a higher vitamin E concentration than the liver. The importance of vitamin E in protecting lung membranes was assessed by using lung and liver isolated from vitamin E-deficient and vitamin E-supplemented rats. For both lung and liver microsomal fractions an inverse relationship between vitamin E concentrations and the extent of lipid peroxidation was observed. However, although the vitamin E concentrations in lung and liver microsomes isolated from rats submitted to a vitamin E-deficient diet were not different, lung microsomes still exhibited a lower production of TBA-RS and ethane than liver. In addition to vitamin E, other factors must be involved to explain the resistance of lung microsomes to lipid peroxidation.

Inhibition of dihydropyridine-sensitive calcium channels by the plant alkaloid ryanodine.

At micromolar concentrations, ryanodine interacts with the dihydropyridine receptor of rabbit skeletal muscle transverse tubules. Ryanodine displaces specifically bound [3H]PN200-110 with an apparent inhibition constant of approx. 95 microM and inhibits dihydropyridine-sensitive calcium channels in the same preparation with an IC50 of approx. 45 microM. These concentrations of ryanodine are approximately three orders of magnitude higher than those required to saturate binding of the alkaloid to the ryanodine receptor of sarcoplasmic reticulum and to open the calcium release channel of sarcoplasmic reticulum (i.e. 20 nM (1988) J. Gen. Physiol. 92, 1-26). Thus at sufficiently high dose, ryanodine may affect SR as well as plasma membrane Ca permeabilities.

Effects of carbohydrates on membrane stability at low water activities.

The relative effectiveness of a variety of carbohydrates in preserving the structural and functional integrity of membranes at low water activities was studied, using Ca-transporting microsomes from muscle as a model membrane. The order of effectiveness (greatest to lowest) was: trehalose, lactose, maltose, cellobiose, sucrose, glucose, fructose, sorbitol, raffinose, myo-inositol, glycerol. At the highest concentrations of the most effective sugars tested, microsomes were obtained upon rehydration that were similar structurally and functionally to fresh membranes. The least effective carbohydrates, alcohol sugars, all appear to be fusogenic. A structural explanation for relative effectiveness of the sugars was sought, but no clear relationship was found, except that effectiveness does not appear to be related to the number of position of hydroxyl groups available for hydrogen bonding.

Subcellular hypothalamic fractions active on ovulation and luteinizing hormone release in the rat.

Nuclear, microsomal and cytosol fractions were obtained from hypothalamic cells by differential centrifugation in sucrose solutions of different molarities. The nuclear fraction (NF) from immature female rats had an inhibitory effect on ovulation induced with pregnant mare's serum (PMS) in immature rats and with luteinizing hormone (LH) in chlorpromazine (CPZ)-treated proestrous rats. The microsomal fraction from the same rats increased both types of ovulation. Nuclear and microsomal fractions obtained from immature male rats were inactive on ovulation. Cytosol fractions were inactive. NFs active to inhibit ovulation significantly reduced the release of LH induced with synthetic LH-RH in immature male rats and in chronically castrated male rats primed with testosterone (T).