Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD levels and were reversed by inhibition of NAD synthesis. 78c increased NAD levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD decline and subsequent metabolic dysfunction.

Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.

Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales, such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provide a dynamic system for a cell to respond to changing needs of Ca2+ regulation.

To understand how the plasma membrane Ca 2؉ pump (PMCA) behaves under changing Ca 2؉ concentrations, it is necessary to obtain information about the Ca 2؉ dependence of the rate constants for calmodulin activation (k act ) and for inactivation by calmodulin removal (k inact ). Here we studied these constants for isoforms 2b and 4b. We measured the ATPase activity of these isoforms expressed in Sf9 cells. For both PMCA4b and 2b, k act increased with Ca 2؉ along a sigmoidal curve. At all Ca 2؉ concentrations, 2b showed a faster reaction with calmodulin than 4b but a slower off rate. On the basis of the measured rate constants, we simulated mathematically the behavior of these pumps upon repetitive changes in Ca 2؉ concentration and also tested these simulations experimentally; PMCA was activated by 500 nM Ca 2؉ and then exposed to 50 nM Ca 2؉ for 10 to 150 s, and then Ca 2؉ was increased again to 500 nM. During the second exposure to 500 nM Ca 2؉ , the activity reached steady state faster than during the first exposure at 500 nM Ca 2؉ . This memory effect is longer for PMCA2b than for 4b. In a separate experiment, a calmodulin-binding peptide from myosin light chain kinase, which has no direct interaction with the pump, was added during the second exposure to 500 nM Ca 2؉ . The peptide inhibited the activity of PMCA2b when the exposure to 50 nM Ca 2؉ was 150 s but had little or no effect when this exposure was only 15 s. This suggests that the memory effect is due to calmodulin remaining bound to the enzyme during the period at low Ca 2؉ . The memory effect observed in PMCA2b and 4b will allow cells expressing either of them to remove Ca 2؉ more quickly in subsequent spikes after an initial activating spike.There are only two mechanisms that actively extrude Ca 2ϩ from the cytosol to the extracellular space: the Na ϩ /Ca 2ϩ exchanger and the plasma membrane Ca 2ϩ pump (PMCA). 1 From comparison of the sequence of PMCA with the x-ray structure of the homologous sarco/endoplasmic reticulum Ca 2ϩ -ATPase(1), it is accepted that its basic structure contains 10 transmembrane domains and two large cytosolic loops. The larger cytosolic loop contains the sites for ATP and formation of the phosphorylated intermediate. In addition, after the last transmembrane domain, PMCAs have a cytoplasmic C-terminal region that includes the calmodulin-binding domain. Although there is strong evidence supporting the formation of oligomers of PMCA when this protein has been solubilized with detergents and purified to a high concentration (2), there is no evidence that these oligomers form when the pump is in the plasma membrane. There are four genes encoding PMCAs; the resulting isoforms are accordingly named PMCA 1, 2, 3, and 4. The diversity of PMCA isoforms is greatly enhanced by the existence of two alternative splicing sites, denoted A and C. No functional differences have yet been found for the alternative splices at site A. Splicing site C is located in the middle of the calmodulinbinding domain in the C-terminal region of the molec...

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

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A reconstitution system allowed us to measure the ATPase activity of specific isoforms of the plasma membrane Ca 2؉ pump continuously, and to measure the effects of adding or removing calmodulin. The rate of activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 nM calmodulin and 0.5 M free Ca 2؉ ) of about 1 min. The rate of inactivation of isoform 4b when calmodulin was removed was even slower, with a half-time of about 20 min. Isoform 4a has a lower apparent affinity for calmodulin than 4b, but its activation rate was surprisingly faster (half time about 20 s). This was coupled with a much faster inactivation rate, consistent with its low affinity. A truncated mutant of isoform 4b also had a more rapid activation rate, indicating that the downstream inhibitory region of fulllength 4b contributed to its slow activation. The results indicate that the slow activation is due to occlusion of the calmodulin-binding domain of 4b, caused by its strong interaction with the catalytic core. Since the activation of 4b occurs on a time scale comparable to that of many Ca 2؉ spikes, this phenomenon is important to the function of the pump in living cells. The slow response of 4b indicates that this isoform may be the appropriate one for cells which respond slowly to Ca 2؉ signals.Unlike other mechanisms for removing Ca 2ϩ from the cytosol, the plasma membrane Ca 2ϩ pump requires activation by another protein, calmodulin. The requirement for this extra step may have profound effects on the shape of Ca 2ϩ spikes, particularly if the binding of calmodulin to the pump is slow. The activation by calmodulin of several calmodulin-regulated enzymes is fast, but it has been observed that the activation of the plasma membrane Ca 2ϩ pump is slow in human erythrocytes (1). Since the binding of calmodulin to the plasma membrane Ca 2ϩ pump is very tight, this slowness in the activation was surprising. Erythrocytes contain a mixture of isoforms 1 and 4 of the plasma membrane Ca 2ϩ pump (2), so it was not clear which isoform was responsible for the slow activation. It was possible to study the rate of activation in erythrocytes because almost all of their Ca 2ϩ -stimulated ATPase activity is due to the plasma membrane Ca 2ϩ pump, but extension of such studies to other cell types has been difficult. The major difficulty is the presence in almost all kinds of cells of non-pump Ca 2ϩ ATPases whose activity swamps that of the pump. Some studies using Ca 2ϩ indicators in whole cells other than erythrocytes have given results consistent with slow activation of the pump. In human neutrophils (3) it was concluded that a Ca 2ϩ spike was caused by delayed activation of the plasma membrane Ca 2ϩ pump. In this case the arguments were based in part on the use of a calmodulin antagonist, which is rather nonspecific. In vascular endothelial cells (4) a similar conclusion was based on the use of La 3ϩ , VO 4 3Ϫ and Hg 2ϩ as inhibitors of the plasma membrane Ca 2ϩ pump. Each of these reagents also inhibits other pumps a...

In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)