The cysteinyl leukotrienes (leukotriene C4, D4 and E4) have potent biological actions which significantly contribute to the airway obstruction in asthma. Several of these effects are blocked by drugs known as CysLT1-receptor antagonists. However, there are actions of leukotrienes which are not sensitive to these antagonists, suggesting the presence of additional receptor subtypes. It was the aim of this Thesis to extend the knowledge about receptors for cysteinyl leukotrienes. Three different isolated smooth muscle preparations kept in organ baths under non-flow conditions were characterised with respect to responsiveness to cysteinyl leukotrienes and sensitivity to purported CysLT1-receptor antagonists. In addition, the study involved evaluation of a leukotriene E4 analogue, BAY u9773, suggested to inhibit responses which cannot be blocked by CysLT1-receptor antagonists. These responses have provisionally been considered to be mediated by CysLT2-receptors. In the guinea pig ileum, BAY u9773 but not the selective CysLT1 receptor antagonist ICI 198,615 inhibited the contractile response to leukotriene C4 in a fashion suggesting competitive antagonism. In sheep trachealis muscle, BAY u9773 antagonised contractions induced by leukotriene C4 and leukotriene D4 in a similar manner, whereas ICI 198,615 did not. The observations support that leukotriene C4 in guinea pig ileum, and leukotriene C4 as well as leukotriene D4 in sheep trachealis muscle, mediated contractions via activation of CysLT2-receptors. In guinea pig lung parenchyma, the effects of BAY u9773 and conventional cysteinyl leukotriene receptor antagonists (ICI 198,615, FPL 55,712) were more complex. First, BAY u9773 evoked a contraction, which could be inhibited by antagonists of CysLT1- and TP-receptors. This suggested that BAY u9773 acted as an agonist at these two receptors. Second, pretreatment with BAY u9773 inhibited a distinct but relatively small component of the contractile response to leukotriene C4 and D4. The effects of BAY u9773 and ICI 198,615 were similar in guinea pig lung parenchyma. The findings suggest that the receptor mediating the major part of the contractile response to exogenous cysteinyl leukotrienes in guinea pig lung parenchyma was different from the currently defined CysLT2-receptor. Furthermore, the data suggested that BAY u9773 was a partial agonist at cysteinyl leukotriene receptors, which presumably contributed to its profile of activity as a combined CysLT1- and CysLT2-receptor antagonist. In addition to contracting guinea pig lung parenchyma, leukotriene C4 and lipoxin A4 also evoked release of thromboxane A2. This release was sensitive to CysLT1-receptor antagonists and contributed to part of the contractile response. Finally, the investigations included a characterisation of the role of leukotrienes in antigen-induced contractions of lung parenchyma from actively sensitised guinea pigs. Combination of antihistamines with CysLT1-receptor antagonists or inhibitors of leukotriene biosynthesis blocked the major component of the antigen-induced contraction. The findings are similar to those observed in isolated human bronchi and support that this model may be used to investigate mediator mechanisms of relevance to asthma.
Cysteinyl-leukotrienes, i.e. leukotriene (LT) C4, D4 and E4, are inflammatory mediators and potent airway- and vasoconstrictors. Two different cysteinyl-leukotriene receptors have been cloned, CysLT1 and CysLT2. This report reviews recent data on CysLT receptor characterisation as well as studies of modulatory mechanisms involved in cysteinyl-leukotriene-induced responses. On the basis of functional studies in isolated smooth muscle preparations, the existence of an additional receptor for cysteinyl-leukotrienes is suggested. In addition, cysteinyl-leukotriene responses in pulmonary vessels were regulated by the release of modulatory factors, of which cyclooxygenase products dominated in the arteries and nitric oxide was the main modulator in porcine pulmonary veins. Moreover, the interconversion between LTC4 and LTD4 and the metabolism into LTE4 may represent a major modulatory mechanism in the guinea-pig trachea by deciding which CysLT receptor is activated by the cysteinyl-leukotrienes.
ABSTRACTUnderstanding the cellular and molecular adaptations of skeletal muscle in response to strength training is important to provide frameworks to improve performance of elite and recreational athletes as well as the health and quality of life of the general population with or without chronic di
Prostaglandin D2 (PGD2) is the major cyclooxygenase metabolite of arachidonic acid released after stimulation of mast cells. Quantification of metabolites of PGD2 can be used as an objective indices of PGD2 production and hence mast cell activation in vivo. The aim of this thesis was to investigate the feasibility of measuring the primary urinary metabolite of PGD2, 9 alpha,11 beta-PGF2 with enzyme immunoassay (EIA). Measurements of 9 alpha,11 beta-PGF2 in urine made by EIA were compared with values obtained by negative ion chemical ionisation gas chromatography-mass spectrometry (NCI GC-MS), the gold standard method. Levels of 9 alpha,11 beta-PGF2, in urine samples measured by NCI GC-MS were consistently lower than those obtained by EIA. NCI GC-MS analysis revealed the presence of two additional dinor compounds, shorter metabolites of 9 alpha,11 beta-PGF2 in the urine. One of the compounds was identical to 9 alpha,11 beta-2,3-dinor-PGF2 which was generated by beta-oxidation of 9 alpha,11 beta-PGF2 and identified by electron impact (EI GC-MS). Thus, urinary 9 alpha,11 beta-PGF2 concentrations measured by EIA represent the sum of three PGD2 metabolites. For convenience sake, the metabolites are collectively referred to as 9 alpha,11 beta-PGF2 in the subsequent studies. A 3-fold increase in the urinary excretion of 9 alpha,11 beta-PGF2 was documented after allergen-induced bronchoconstriction in nine atopic asthmatics. This challenge was considered a positive control since it is unambiguous that mast cell activation occurs during the early phase of allergen-induced airway obstruction. Histamine-induced bronchoconstriction did not result in an increase in the levels of 9 alpha,11 beta-PGF2 demonstrating that PGD2 was not formed as a consequence of the bronchoconstriction per se. Moreover, bronchial challenge with lysine-aspirin in eight aspirin-intolerant asthmatics elicited bronchoconstriction and was accompanied by a significant increase in the urinary excretion of 9 alpha,11 beta-PGF2. Challenge with a higher dose of aspirin produced an even greater increase in 9 alpha,11 beta-PGF2 levels, indicating a dose-dependent release of PGD2 during aspirin-induced bronchoconstriction. The pattern of mediator release during the early (EAR) and late asthmatic response (LAR) to allergen was investigated by subjecting twelve mild atopic asthmatics to allergen challenge. Within one hour of the maximal bronchoconstrictor response, there was a significant increase in the urinary concentrations of the mast cell markers, 9 alpha,11 beta-PGF2 and N tau-methylhistamine, urinary metabolite of histamine, and the end product of the cysteinyl-leukotrienes, leukotriene (LT)E4. Levels of all three mediators were also significantly elevated above baseline during the LAR. Urinary levels of eosinophil protein X (EPX), a marker of eosinophil activation, remained unaltered during both the EAR and LAR. Preliminary evidence suggests a diurnal variation in the urinary excretion of EPX. Increased airway fluid osmolarity in the lower airways as a result of exercise, has been suggested to trigger mast cell activation and subsequent bronchoconstriction in a subset of asthmatics. Twelve subjects with a history of exercise-induced bronchoconstriction (EIB), exercised on a stationary bicycle ergometer for 5 minutes. Seven of the subjects (responders) experienced bronchoconstriction, whereas, the pulmonary function of the remaining five subjects (non-responders) remained stable. The urinary excretion of 9 alpha,11 beta-PGF2 in the responder group increased significantly compared to the non-responders at 30 and 90 minutes after exercise. The urinary excretion of LTE4 and N tau-methylhistamine was not significantly different between the two groups at either time point after exercise, although there was a tendency for elevated levels of N tau-methylhistamine in the responder group. (ABSTRACT TRUNCATED)
Alanine-scanning mutagenesis of all amino acids in transmembrane helices M4, M5, M6 and M8, which contain known Ca2+ binding residues in the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, revealed patches of mutation-sensitivity in M4, M5 and M6, but in M8. A six-residue motif, (E/D)GLPA(T/V), in M4 and M6 and its counterpart in M5 were highlighted by mutagenesis. Site-directed disulfide mapping of helices M4 and M6 demonstrated that these transmembrane helices associate as a right-handed coiled-coil. This structural information, combined with the earlier analysis of the association of each Ca2+ binding residue with either Ca2+ binding site I or site II, permitted the development of a "side-by-side" model for the two Ca2+ binding sites in the Ca(2+)-ATPase. In about half of Brody disease families, mutations create stop codons which delete all or part of the Ca2+ binding and translocation domain, resulting in loss of SERCA1 function and muscle disease.
The yeast plasma membrane [H+]-ATPase is essential for cell viability, pumping protons across the cell membrane to generate a large electrochemical gradient that provides the energy for nutrient uptake (reviewed by Goffeau amp; Slayman, 1981; Serrano, 1988; and Nakamoto amp; Slayman, 1989). The [H+]-ATPase is encoded by the PMA1 gene (Serrano et al., 1986) and belongs to a widely distributed family of transporters known as E1E2- or P-ATPases. Like other members of the group, it has a 100 kDa catalytic subunit that is firmly embedded in the lipid bilayer (Dufour amp; Goffeau, 1978) and alternates between two major conformational states (Ei and E2), hydrolyzing ATP by way of a covalent B-aspartyl phosphate reaction intermediate (Dame amp; Scarborough, 1981; Amory amp; Goffeau, 1982).
The two Ca2+ pumps of higher eucaryotes are strictly targeted to different membrane systems: the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). Chimeric constructs of the two pumps expressed in COS-7 cells have revealed a strong signal for endoplasmic reticulum retention in the N-terminal cytosolic portion of the SERCA pump: the signal is contained in a stretch of 28 amino acids that follows the N-terminus. A second, but masked, endoplasmic reticulum retention signal is contained in a cytosolic C-terminal sequence immediately preceding the calmodulin-binding domain of the Ca2+ pump. Selective mutations on the SERCA pump have led to the conclusion that 5 conserved residue membrane domains (TM)4, 5, and 6 form the Ca2+ channel through the pump protein. A comparative sequence inspection has failed to reveal any of these residues in TM5 of the PMCA pump. Mutation of the conserved residue in TM4 and of two in TM6 abolished the ability of the pump to form the Ca(2+)-dependent phosphoenzyme. However, one of the mutations (N979, TM6) also caused retention of the PMCA pump in the reticulum, suggesting structural alterations. Of the four basic isoforms of the pump, two (1, 4) are ubiquitously expressed, two (2, 3) are essentially brain specific. Isoform 2 has the highest calmodulin affinity. Primary cultures of cerebellar granule cells from newborn rats did not express isoforms 2 and 3 at plating time. Incubation of the cells in depolarizing concentrations of KCl, which promote Ca2+ influx, promoted the expression of isoforms 2 and 3, and of a brain specific spliced variant of isoform 1. Incubation of the cells in L-type Ca2+ channel blockers abolished the upregulation of the pump genes.
The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.
Our studies have concentrated on two aspects of the Na,K-ATPase, the first relates to the identification of amino acids involved in binding Na+ and K+ during the catalytic cycle and the second involves defining how cardiac glycosides inhibit the enzyme. To date, three amino acids, Ser775, Asp804 and Asp808, all located in transmembrane regions five and six, have been shown to play a major role in K+ binding. These findings are based on site directed mutagenesis and expression studies. In order to understand how cardiac glycosides interact with the Na,K-ATPase, studies again involving mutagenesis coupled with expression have been used. More specifically, amino acid residues have been substituted in an ouabain sensitive alpha subunit using random mutagenesis, and the ability of the resulting enzyme to confer resistance to ouabain sensitive cells was determined. Interestingly, the amino acids of the alpha subunit which alter ouabain sensitivity cluster in two major regions, one comprised of the first and second transmembrane spanning domains and the extracellular loop joining them, and the second formed by the extracellular halves of transmembrane regions four, five, six and seven. As noted above, transmembrane regions five and six also contain the three amino acid residues Ser775, Asp804 and Asp808 which play a key role in cation transport, possibly binding K+. Thus, it is reasonable to propose that cardiac glycosides bind to two sites, the N- terminal region and the central region which contains the cation binding sites. Cardiac glycoside binding to the center region may lock the cation transport region into a configuration such that the enzyme cannot go through the conformational change required for ion transport.
This paper describes a novel technique for specific cleavage of renal Na/K-ATPase, based on bound transition metal ions. The approach might have application to other P-type pumps or membrane proteins. In one type of experiment, specific cleavages of the alpha subunit have been observed following incubation with ascorbate plus H2O2. Five fragments with intact C-terminals and complementary fragments with intact N-terminals are detectable. The beta subunit is not cleaved. Cleavages depend on the presence of contaminant or added submicromolar concentrations of Fe2+ ions. The results suggest that Fe2+ (or Fe3+) binds with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. The rate of cleavage is greatly affected by the conformational state of the protein, E1Na or E2(Rb), respectively. The findings provide information on spatial organization of the protein and suggest that the highly conserved regions of the alpha subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations, but move apart in the E1 or E1Na conformations. In a second application of this technique, added Cu2+ ions at micromolar concentrations, have been shown to catalyse specific cleavages of both alpha and beta subunits at the extracellular surface. The experiments provide evidence for trans-membrane topology and proximity between trans-membrane segments M5-M10 within the alpha subunit and for interacting segments of alpha and beta subunits. We discuss the implications of metal-catalysed cleavages for spatial organisation of transmembrane helices of the protein.
The 5,885 members of the yeast proteome have been screened for amino acid sequence signatures of either P-type ATPases or ABC transporters. A total of 16 P-type ATPases have been classified into six phylogenetic families which each seem to transport a specific class of substrates. In addition, a total of 16 ABC transporters comprising two nucleotide binding folds and two membrane domains were classified in two distinct phylogenetic families. Two ABC transporters of Family I (Pdr5p and Snq2p) share overlapping promiscuity for numerous hydrophobic drugs with a member of Family II (Yor1p). In this case, substrate specificity seems to have differentiated more slowly during evolution than typical phylogenetic traits reflected by amino acid sequence similarity or predicted membrane topography.
The kdpFABC operon of Escherichia coli consists of the four structural genes kdpF, kdpA, kdpB, and kdpC. Expression of the kdpF gene was demonstrated using minicells of E. coli. In addition, it was shown that the KdpF subunit remains associated with the purified complex. Although KdpF is not essential in vivo, the purified complex lacking KdpF exhibits hardly any K(+)-stimulated ATPase activity. This clearly demonstrates that the KdpF subunit is stabilizing the transport complex. Charge translocation by the purified Kdp-ATPase was measured with the potential-sensitive dye DiSC3(5) using proteoliposomes. Upon addition of ATP a fluorescence quench was observed indicating the buildup of a negative potential inside the proteoliposomes. Using the Kdp-ATPase derived from a mutant strain, in which the K(m) value for K+ (1,2 mM) was almost identical to that of Rb+ (1.4 mM), the same fluorescence quench was observed when K+ or Rb+ were present in the lumen of the proteoliposomes. These data clearly indicate that the Kdp-ATPase transports K+ in an electrogenic manner. In order to identify the binding site(s) for the inhibitor concanamycin A within the Kdp complex, concanamycin A was synthesized. Using this compound labeling of KdpA and KdpB, but not of KdpC, could be shown with the purified complex. When everted vesicles were used only KdpB could be labeled.
The ATP synthase F1F0 is the smallest molecular motor yet studied. ATP hydrolysis drives the rotary motion of the primary stalk subunits gamma and epsilon relative to the alpha 3 beta 3 part of F1. Evidence is reviewed to show that the delta and b subunits provide a second stalk that can act as a stator to facilitate these rotational movements.
Cotransporters are a major class of membrane transport proteins that are responsible for the accumulation of nutrients, neurotransmitters, osmolytes and ions in cells from bacteria to man. The energy for solute accumulation comes from the proton and/or sodium electrochemical gradients that exist across cell membranes. A major problem in biology is how transport is coupled to these electrochemical potential gradients. The primary example of this class of membrane proteins is the intestinal brush border Na+/glucose cotransporter (SGLT1), first described by Bob Crane in 1960. Over 35 members of the SGLT1 gene family have been identified in animal cells, yeast and bacteria, and all share a common core structure of 13 transmembrane (TM) helices. Electrophysiological techniques have been used to examine the function of several family members, chimeras and mutants expressed in heterologous systems such as Xenopus laevis oocytes. These have revealed that cotransporters are multi-functional proteins: they are responsible for 1). uncoupled passive Na+ transport (Na+ uniport); 2). down-hill water transport in the absence of substrate; 3). Na+/substrate cotransport; and 4). Na+/substrate/water cotransport. The sugar binding and translocation pathway is formed by 4 TM helices near the C-terminal of the protein, helices 10-13. We propose that the N-terminal domains of SGLT1 are responsible for Na+ binding and/or translocation, and that Na+/glucose cotransport results from interactions between the N- and C-terminal domains of the protein.
The gene defective in cystic fibrosis encodes a Cl- channel named CFTR, which belongs to the family of transport proteins identified by their cytoplasmic domains that bind and hydrolyse ATP. CFTR channels require phosphorylation by protein kinase A at one or more serine residues in the large central regulatory domain before they will open. Severl findings argue that hydrolysis of ATP at the N-terminal nucleotide binding domain is the rate-limiting step for opening a phosphorylated CFTR channel. Although AMP-PNP the non-hydrolysable, but close structural, analog of ATP fails to open phosphorylated CFTR channels, once a channel has been opened, AMP-PNP can bind tightly to the channel and "lock" it into the open conformation for several minutes. This tight binding of AMP-PNP presumably occurs at CFTR's C-terminal nucleotide binding domain. Because it structurally resembles AMP-PNP, ATP must also bind tightly there, which suggests that hydrolysis of that ATP normally prompts channel closing. That conclusion is supported by the finding that free [Mg2+] level controls the rate of CFTR channel closure. A normal closed-open-closed gating cycle of a CFTR channel thus seems to involve hydrolysis of one ATP molecule to open it, and hydrolysis of a second ATP to close it. Stabilization of an active state by tight binding of a nucleotide, and termination of that state by hydrolysis of the nucleotide, are characteristics reminiscent of G proteins. Indeed, CFTR's nucleotide binding domains share with G proteins not only this functional similarity, but also some sequence homology, at least in certain highly conserved motifs.
