The synthesis of a small heat shock/alpha-crystallin protein in Artemia and its relationship to stress tolerance during development.
(1/97)
Fertilized oocytes of the brine shrimp Artemia franciscana undergo either ovoviviparous or oviparous development, yielding free-swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Encystment is followed by diapause, wherein metabolism is greatly reduced; the resulting cysts are very resistant to extreme stress, including desiccation and long-term anoxia. The synthesis of p26, a small heat shock/alpha-crystallin protein produced only in oviparously developing Artemia, is shown in this paper to be transcriptionally regulated. A p26 mRNA of about 0.7 kb was detected on Northern blots in the second day after oocyte fertilization. It peaked as embryos encysted and declined rapidly when activated cysts resumed development. The appearance of p26 protein, as indicated by immunoprobing of Western blots, followed mRNA by 1 day; it also increased as encystment occurred but remained constant during postgastrula development of cysts. However, p26 underwent a marked reduction during emergence of nauplii and could not be detected in cell-free extracts of second-instar larvae. p26 entered nuclei of encysting embryos soon after synthesis and was localized therein as late as instar II, when it was restricted to a small set of salt gland nuclei. First-instar larvae derived from cysts were more thermotolerant than larvae that had developed ovoviviparously, but synthesis of p26 was not induced by heat under the experimental conditions employed. Additionally, transformed bacteria synthesizing p26 were more thermotolerant than bacteria that lacked the protein. The results support the proposal that p26, a developmentally regulated protein synthesized during embryo encystment, has chaperone activity in vivo and protects the proteins of encysted Artemia from stress-induced denaturation. (+info)
Mecamylamine blocks the [Asp1,Val5]-ANG II-induced attenuation of salt gland activity in Pekin ducks.
(2/97)
An intravenous injection of 2 microg of [Asp1,Val5]-ANG II attenuated fluid secretion by the nasal salt glands of Pekin ducks. Ganglionic blockade with mecamylamine stopped salt gland secretion. Flow was reestablished by intravenous methacholine bromide during ganglionic blockade. A second injection of 2 microg of [Asp1, Val5]-ANG II failed to attenuate secretion during ganglionic blockade, showing that the peptide acts via the central nervous system and postganglionic parasympathetic nerves that supply the salt glands. Sympathetic nerves are located in the walls of blood vessels within the salt glands, and adrenergic fibers with "varicosities" supply extensively the secretory tubules. [Asp1, Val5]-ANG II decreased salt gland secretion both before and after alpha1-adrenergic blockade with prazosin, showing that the lowered activity was not caused by the release of norepinephrine from nerve endings and/or duck adrenal chromaffin cells. beta-Adrenergic blockade with propranolol also failed to prevent the attenuation of secretion in response to an intravenous injection of 2 microg of [Asp1,Val5]-ANG II, which showed that epinephrine did not mediate the response to the peptide. (+info)
APH-1, a POU homeobox gene expressed in the salt gland of the crustacean Artemia franciscana.
(3/97)
We characterized the first POU-homeoprotein in a crustacean (designated APH-1 for Artemia POU-Homeoprotein, EMBL Y15070). The amino acid sequence of the APH-1 POU-domain is identical, except for two residues, to that of the two class III POU proteins Cf1-a (Drosophila) and POU-M1 (Bombyx mori). Southern blot analysis suggests that crustaceans have only one class III POU gene. RT-PCR and whole-mount in situ hybridization show that APH-1 mRNA is present in larvae specifically in the salt gland, an organ which is involved in osmoregulation, and disappears in the adult. (+info)
Mode of activation of salt secretion by C-type natriuretic peptide in the shark rectal gland.
(4/97)
We studied the modes of activation of the salt-secreting rectal gland of the spiny dogfish, Squalus acanthias, by the native cardiac peptide CNP. The stimulatory action of CNP in isolated perfused glands is inhibited by 10 mM procaine, presumably by blocking release of vasoactive intestinal peptide (VIP) from nerves. Procaine reduces the slope of the dose-response curve of human CNP and that of shark CNP (each P < 0.0001). CNP increases short-circuit current in cultured rectal gland cells from 4.8 +/- 1.6 to 27.0 +/- 7.8 microA/cm2. It also stimulates the secretion of chloride in isolated perfused glands in the presence of 10 mM procaine from 72 +/- 31 to 652 +/- 173 microeq. h(-1). g(-1). These results suggest that CNP has a direct cellular action not mediated by the neural release of VIP. The residual stimulation of perfused glands in the presence of procaine was almost completely inhibited by staurosporine [10 nM; an inhibitor of protein kinase C (PKC)] from 652 +/- 173 to 237 +/- 61 microeq. h(-1). g(-1). Although CNP stimulates guanylyl cyclase in shark rectal gland, chloride secretion of perfused glands was not elicited by 8-bromoadenosine-cGMP (8-BrcGMP) alone nor by the activator of PKC phorbol ester. The combination of PKC activation and 8-BrcGMP infusion, however, stimulated chloride secretion in perfused glands from 94 +/- 30 to 506 +/- 61 microeq. h(-1). g(-1), a level comparable to that observed in glands blocked with procaine. Several parallel pathways appear to be synergistic in activating chloride secretion stimulated by CNP in the rectal gland. (+info)
Alphabeta protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: formation of higher oligomers does not modify molecular activity.
(5/97)
The distance that separates alphabeta protomers of the Na(+), K(+)-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of alphabeta protomers and either N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 A farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism. (+info)
Ca(2+) and p38 MAP kinase regulate mAChR-mediated c-Fos expression in avian exocrine cells.
(6/97)
Muscarinic acetylcholine receptors (mAChRs) in exocrine tissue from the avian nasal salt gland are coupled to phospholipase C and generate inositol phosphate and Ca(2+) signals upon activation. An early effect of receptor activation in the secretory cells is a transient accumulation of c-Fos protein. In cooperation with constitutively expressed Jun, Fos presumably serves as a transcription factor altering gene expression during cell growth and differentiation processes in the gland associated with adaptation to osmotic stress in animals. Nothing is known, however, about the mAChR-dependent signaling pathways leading to Fos expression in these cells. By incubation of isolated nasal gland tissue in short-term culture with activators or inhibitors of signaling pathways and quantitative Western blot analysis of Fos abundance, we have now identified the sustained elevation of the intracellular Ca(2+) concentration and the activation of the p38 mitogen-activated protein (MAP) kinase as intermediate signaling elements for the regulation of c-Fos by muscarinic receptor activation. It is suggested that p38 MAP kinase, rather than exclusively mediating stress responses, is involved in the regulation of cellular growth and differentiation controlled by G protein-coupled receptors. (+info)
Ligands presumed to label high affinity and low affinity ATP binding sites do not interact in an (alpha beta)2 diprotomer in duck nasal gland Na+,K+-ATPase, nor Do the sites coexist in native enzyme.
(7/97)
The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same alphabeta protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each alphabeta protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITC-bound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITC-labeled enzyme, TNP-ADP and ErITC inhibit p- nitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC. (+info)
Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase c via a novel member of the FXYDY family.
(8/97)
The Na,K-ATPase provides the driving force for many ion transport processes through control of Na(+) and K(+) concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, alpha and beta. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the gamma-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the gamma-subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na, K-ATPase alpha-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the alpha-subunit of pig kidney Na, K-ATPase, indicating specific association with different alpha-isoforms. Finally, PLMS and the alpha-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C(12)E(8) resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase. (+info)