RESUMO
The excretion of nitrogenous waste products in the form of ammonia (NH3) and ammonium (NH4 (+)) is a fundamental process in aquatic organisms. For mytilid bivalves, little is known about the mechanisms and sites of excretion. This study investigated the localization and the mechanisms of ammonia excretion in mytilid mussels. An Rh protein was found to be abundantly expressed in the apical cell membrane of the plicate organ, which was previously described as a solely respiratory organ. The Rh protein was also expressed in the gill, although at significantly lower concentrations, but was not detectable in mussel kidney. Furthermore, NH3/NH4 (+) was not enriched in the urine, suggesting that kidneys are not involved in active NH3/NH4 (+) excretion. Exposure to elevated seawater pH of 8.5 transiently reduced NH3/NH4 (+) excretion rates, but they returned to control values following 24â h acclimation. These mussels had increased abundance of V-type H(+)-ATPase in the apical membranes of plicate organ cells; however, NH3/NH4 (+) excretion rates were not affected by the V-type H(+)-ATPase specific inhibitor concanamycin A (100â nmolâ l(-1)). In contrast, inhibition of ciliary beating with dopamine and increased seawater viscosity significantly reduced NH3 excretion rates under control pH (8.0). These results suggest that NH3/NH4 (+) excretion in mytilid mussels takes place by passive NH3 diffusion across respiratory epithelia via the Rh protein, facilitated by the water current produced for filter feeding, which prevents accumulation of NH3 in the boundary layer. This mechanism would be energy efficient for sessile organisms, as they already generate water currents for filter feeding.
Assuntos
Amônia/metabolismo , Bivalves/metabolismo , Cílios/metabolismo , Estruturas Animais/anatomia & histologia , Estruturas Animais/enzimologia , Animais , Bivalves/enzimologia , Epitélio/metabolismo , Brânquias/metabolismo , Hemolinfa/metabolismo , Concentração de Íons de Hidrogênio , Transporte de Íons , Proteínas/metabolismo , Água do Mar/química , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (TAL) is stimulated by AVP via V2 receptor/PKA/cAMP signaling. This process is antagonized by locally produced eicosanoids such as 20-HETE or prostaglandin E(2), which are synthesized in a phospholipase A(2)-dependent reaction cascade. Using microarray-based gene expression analysis, we found evidence for an AVP-dependent downregulation of the calcium-independent isoform of PLA(2), iPLA(2)ß, in the outer medulla of rats. In the present study, we therefore examined the contribution of iPLA(2)ß to NKCC2 regulation. Immunoreactive iPLA(2)ß protein was detected in cultured mTAL cells as well as in the entire TAL of rodents and humans with the exception of the macula densa. Administration of the V2 receptor-selective agonist desmopressin (5 ng/h; 3 days) to AVP-deficient diabetes insipidus rats increased outer medullary phosphorylated NKCC2 (pNKCC2) levels more than twofold in association with a marked reduction in iPLA(2)ß abundance (-65%; P < 0.05), thus confirming microarray results. Inhibition of iPLA(2)ß in Sprague-Dawley rats with FKGK 11 (0.5 µM) or in mTAL cells with FKGK 11 (10 µM) or (S)-bromoenol lactone (5 µM) for 1 h markedly increased pNKCC2 levels without affecting total NKCC2 expression. Collectively, these data indicate that iPLA(2)ß acts as an inhibitory modulator of NKCC2 activity and suggest that downregulation of iPLA(2)ß may be a relevant step in AVP-mediated urine concentration.
Assuntos
Fosfolipases A2 do Grupo VI/metabolismo , Alça do Néfron/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Vasopressinas/metabolismo , Animais , Anticorpos , Ácidos Araquidônicos , Células Cultivadas , Desamino Arginina Vasopressina , Regulação para Baixo , Fluorocarbonos , Expressão Gênica , Fosfolipases A2 do Grupo VI/imunologia , Cobaias , Humanos , Isoenzimas , Cetonas , Medula Renal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftalenos , Organofosfonatos , Fosforilação , Pironas , Ratos , Ratos Brattleboro , Ratos Sprague-Dawley , Membro 1 da Família 12 de Carreador de SolutoRESUMO
The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca(2+)-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca(2+)-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM-Sepharose in the presence of Ca(2+), and (2) TRP4-glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688-759 and 786-848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca(2+) concentrations above 10 microM, with half maximal binding occurring at 16.6 microM (domain 1) and 27.9 microM Ca(2+) (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694-728 and 829-853, interacted directly with dansyl-CaM with apparent K(d) values of 94-189 nM. These results indicate that TRP4/Ca(2+)-CaM are parts of a signalling complex involved in agonist-induced Ca(2+) entry.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio/química , Proteínas de Ligação a Calmodulina/química , Bovinos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPCRESUMO
Ca2+ influx through high voltage activated Ca2+ channels initiates a number of physiological processes including e.g. excitation-contraction coupling in cardiac myocytes and excitation-transcription coupling in neurones. The Ca2+ channels involved are complexes of a pore-forming alpha1 subunit, a transmembrane delta subunit disulfide-linked to an extracellular alpha2 subunit, a intracellular beta subunit and, at least in some tissues, a gamma subunit. Experimental analysis of beta subunit function comprises functional coexpression of its cDNA together with the cDNAs of the other subunits. This experimental approach can be supplemented by investigating functional alterations that result from the genetic elimination of Ca2+ channel beta genes in mice. Here we summarize the phenotype of mice deficient in the beta1 subunit, the beta3 subunit or the beta4 subunit, respectively.
Assuntos
Canais de Cálcio/genética , Mutação , Animais , CamundongosRESUMO
Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.
Assuntos
Córtex Suprarrenal/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions , Ativação do Canal Iônico , Receptores de Superfície Celular/metabolismo , Córtex Suprarrenal/citologia , Animais , Canais de Cálcio/genética , Bovinos , DNA Antissenso/farmacologia , Condutividade Elétrica , Hibridização In Situ , Inositol 1,4,5-Trifosfato/farmacologia , Dados de Sequência Molecular , RNA Mensageiro/isolamento & purificação , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPC , Tapsigargina/farmacologia , Distribuição TecidualRESUMO
In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.