Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α 9 β 1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N 1 -acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α 9 β 1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α 9 β 1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α 9 β 1 or treated with integrin α 9 β 1 -blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α 9 β 1 . Therefore, integrin α 9 β 1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.
Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT2 encodes a protein 73% identical in amino acid sequence to BHMT, but the function of BHMT2 remains unclear. We set out to identify and functionally characterize common genetic variation in BHMT and BHMT2. Specifically, we sequenced exons, exon-intron splice junctions and the 5′-flanking regions (5′-FRs) of BHMT and BHMT2 using 240 DNA samples from four ethnic groups. Twenty-five single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs, and 39 SNPs, including 4 nonsynonymous, were observed in BHMT and BHMT2, respectively. BHMT wild type (WT) and variant allozymes were expressed in COS-1 cells. Variant allozymes showed no significant differences from WT in levels of enzyme activity or immunoreactive protein, but there were statistically significant differences in apparent K m values. Luciferase reporter gene constructs were created for the three most common BHMT 5′-FR haplotypes, and significant variation was observed in the ability of these constructs to drive transcription. Although BHMT2 mRNA has been observed in human liver and kidney, expression of the protein has not been reported. We were unable to express BHMT2 in mammalian cells, and the protein aggregated after bacterial expression. Furthermore, BHMT2 was rapidly degraded in a rabbit reticulocyte lysate, but it could be stabilized by cotransfection of COS-1 cells with BHMT and, after cotransfection, it coprecipitated with BHMT. These studies have defined common genetic variation in BHMT and BHMT2 and functionally characterized BHMT SNPs. They may also help to explain why BHMT2 has not previously been defined functionally.
Alternative RNA splicing may provide unique opportunities to identify drug targets and therapeutics. We identified an alternative spliced transcript for B-type natriuretic peptide (BNP) resulting from intronic retention. This transcript is present in failing human hearts and is reduced following mechanical unloading. The intronretained transcript would generate a unique 34 amino acid (aa) carboxyl terminus while maintaining the remaining structure of native BNP. We generated antisera to this carboxyl terminus and identified immunoreactivity in failing human heart tissue. The alternatively spliced peptide (ASBNP) was synthesized and unlike BNP, failed to stimulate cGMP in vascular cells or vasorelax preconstricted arterial rings. This suggests that ASBNP may lack the dose-limiting effects of recombinant BNP. Given structural considerations, a carboxyl-terminal truncated form of ASBNP was generated (ASBNP.1) and was determined to retain the ability of BNP to stimulate cGMP in canine glomerular isolates and cultured human mesangial cells but lacked similar effects in vascular cells. In a canine-pacing model of heart failure, systemic infusion of ASBNP.1 did not alter mean arterial pressure but increased the glomerular filtration rate (GFR), suppressed plasma renin and angiotensin, while inducing natriuresis and diuresis. Consistent with its distinct in vivo effects, the activity of ASBNP.1 may not be explained through binding and activation of NPR-A or NPR-B. Thus, the biodesigner peptide ASBNP.1 enhances GFR associated with heart failure while lacking the vasoactive properties of BNP. These findings demonstrate that peptides with unique properties may be designed based on products of alternatively splicing.vasoactive ͉ myocardial ͉ kidney G enome-wide analyses have revealed the prevalence of alternative splicing of multiexonic genes (1, 2). In fact, much of the complexity of the human proteome is accounted for by alternative splicing of messenger RNA. Identification of these altered forms may allow for unique opportunities to diagnose, understand, and treat human disease. Therefore, we hypothesized that it might be possible to identify splice variants and to design therapeutics based on their unique structure and function.As an example of the wide potential of this technology to alter disease states, we focused on a peptide with broad mechanistic, diagnostic, and therapeutic importance in cardiovascular disease, B-type natriuretic peptide (BNP). BNP is encoded by a small multiexonic gene, and although discovered in brain (3), is expressed primarily in the heart (4). BNP, like atrial natriuretic peptide and C-type natriuretic peptide, is expressed as a prepro-hormone that is processed to a mature [32-amino acid (aa)] form by extracellular proteases (5). Mature BNP contains short carboxyl and amino termini and a central 17-aa ring. BNP has important autocrine, paracrine, and endocrine actions that are mediated through the NPRA receptor and activation of cGMP in target cells (6). Infusion of a recombinant form of mat...
SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has recently been described as a potent and selective inhibitor of Na ϩ -Ca 2ϩ exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of SEA0400 were investigated on the cloned cardiac Na ϩ -Ca 2ϩ exchanger, NCX1.1, expressed in Xenopus laevis oocytes to gain insight into its inhibitory mechanism. Na ϩ -Ca 2ϩ exchange currents were measured using the giant excised patch technique using conditions to evaluate both inward and outward currents. SEA0400 inhibited outward Na ϩ -Ca 2ϩ exchange currents with high affinity (IC 50 ϭ 78 Ϯ 15 and 23 Ϯ 4 nM for peak and steady-state currents, respectively). Considerably less inhibitory potency (i.e., micromolar) was observed for inward currents. The inhibitory profile was reexamined after proteolytic treatment of excised patches with ␣-chymotrypsin, a procedure that eliminates ionic regulatory mechanisms. After this treatment, an IC 50 value of 1.2 Ϯ 0.6 M was estimated for outward currents, whereas inward currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400 on outward exchange currents were evident at both high and low concentrations of regulatory Ca 2ϩ , although distinct features were noted. SEA0400 accelerated the inactivation rate of outward currents. Based on paired pulse experiments, SEA0400 altered the recovery of exchangers from the Na ϩ i -dependent inactive state, particularly at higher regulatory Ca 2ϩ i concentrations. Finally, the inhibitory potency of SEA0400 was strongly dependent on the intracellular Na ϩ concentration. Our data confirm that SEA0400 is the most potent inhibitor of the cardiac Na ϩ -Ca 2ϩ exchanger described to date and provide a reasonable explanation for its apparent transport mode selectivity.
The cardiac sodium-calcium exchanger (NCX) plays an important role in calcium homeostasis. It is the primary mechanism for removing calcium ions that enter myocytes through L-type calcium channels on a beat-to-beat basis. Its direction of transport is determined by the membrane potential and the ionic concentrations of Na + and Ca 2+ , with the forward (or Ca 2+ -efflux) mode of transport being the dominant mode under physiological conditions. In contrast, the Ca 2+ -influx mode (or reverse mode) of NCX becomes important in certain pathophysiological conditions, such as myocardial ischemia and reperfusion. Recent discovery of compounds that inhibit the Ca 2+ -influx mode (or reverse mode) of NCX has generated intense research interest in the pharmacology of NCX. Among the newer NCX inhibitors described to date, 2-[4-[(2,5-difluorophenyl)methoxy]-phenoxy]-5-ethoxyaniline (SEA0400) appears particularly promising in attenuating cardiac, renal, and cerebral ischemia/reperfusion injuries in various experimental models. Moreover, the mixed results that have emerged from clinical trials evaluating the efficacy and safety of inhibitors of the sodium-hydrogen exchanger (an upstream target in relation to the sodium-calcium exchanger) in myocardial protection stimulated interest in evaluating NCX as an alternative therapeutic target. This article reviews the pharmacological profile of SEA0400, as presented in the published literature, and discusses the therapeutic potential of this compound in attenuating myocardial ischemia/reperfusion injury.
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