Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ∼90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60–70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition

Enhanced Ca2+-sensing Receptor Function in Idiopathic Pulmonary Arterial Hypertension

Rationale: A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca2+]cyt and enhanced Ca2+ influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Objective: We examined whether the extracellular Ca2+-sensing receptor (CaSR) is involved in the enhanced Ca2+ influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results: In normal PASMC superfused with Ca2+-free solution, addition of 2.2 mM Ca2+ to the perfusate had little effect on [Ca2+]cyt. In IPAH-PASMC, however, restoration of extracellular Ca2+ induced a significant increase in [Ca2+]cyt. Extracellular application of spermine also markedly raised [Ca2+]cyt in IPAH-PASMC, but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca2+-induced [Ca2+]cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca2+-mediated [Ca2+]cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. Conclusions: The extracellular Ca2+-induced increase in [Ca2+]cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca2+ influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension

Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness

Upregulated pro-LOX expression in PASMC isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) is associated with increased stiffness.

<p>A: Western blot analysis on pro-LOX in PASMC from normal subjects (Normal) and IPAH patients (IPAH). B: Time courses of the membrane deformation in normal PASMC (open circles) and IPAH-PASMC (closed circles) cultured on uncoated cover slips. Microaspiration was used to determine the membrane deformation which is inversely related to membrane stiffness. <i>P</i><0.01 between the two curves (using two-way ANOVA). C: Time courses of the membrane deformation in normal PASMC and IPAH-PASMC cultured on collagen-coated cover slips. <i>P</i><0.01 between the two curves (using two-way ANOVA). D: Time courses of the membrane deformation in IPAH-PASMC treated with (IPAH+βAPN) or without (IPAH) the LOX inhibitor βAPN. <i>P</i><0.01 between the two curves (using two-way ANOVA).</p

Downregulation of HIF-1α by siRNA significantly attenuates mRNA expression of CTR1 in hypoxic PASMC.

<p>A: Real-time RT-PCR analysis on HIF-1α (<i>a</i>), HIF-2α (<i>b</i>) and CTR1 (<i>c</i>) in hypoxic PASMC treated with (50–200 pmol) or without (0 pmol) siRNA specifically targeting HIF-1α, HIF-2α and CTR1, respectively. Data are shown in mean±SE. ***<i>P</i><0.01 vs. control hypoxic cells (0-pmol siRNA). B: Real-time RT-PCR analysis on human CTR1 (<i>a</i>), ATP7A (<i>b</i>), HIF-1α (<i>c</i>) and HIF-2α (<i>d</i>) in hypoxic PASMC treated with 100-pmol scrambled siRNA (Control-siRNA, open bars), HIF-1α-siRNA (solid bars), HIF-2α-siRNA (light grey bars), and CTR1-siRNA (dark grey bars), respectively. ***<i>P</i><0.01 vs. hypoxic cells treated with scrambled siRNA (Control-siRNA).</p

Hypoxia-mediated upregulation of mRNA expression of Cu transporters (CTR1, ATP7A) and lysyl oxidase (LOX) is associated with an increase in Cu transportation in human pulmonary arterial smooth muscle cells (PASMC).

<p>A: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and ⁶⁴Cu uptake (mean±SE) in human PASMC exposed to normoxia (Nor) and hypoxia (Hyp, 3% O₂ for 48 hrs, n = 3; right panel). B: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and ⁶⁴Cu uptake (mean±SE, right panel) in human PASMC treated with vehicle (Cont) and CoCl₂ (100 µM for 48 hrs, n = 3; right pane). Lactate dehydrogenase (LDH) and erythropoietin (EPO) were used as positive controls. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. Hyp or CoCl₂.</p

PASMC motility is dependent on Cu and the Cu-dependent PASMC motility is augmented by hypoxia.

<p>Cell motility was determined by a scratch wound assay over a period of 12: Representative images showing normoxic (Nor) and hypoxic (Hyp) PASMC immediately (0 hr) or 12 hrs after scratch with a sterile pipette in the absence (Control) or presence (BCS) of 200 µM BCS (a Cu chelator). B: Representative images showing control (-CoCl₂) and 100-µm CoCl₂-treated PASMC immediately (0 hr) or 12 hrs after scratch in the absence (Control) or presence (BCS) of BCS. C: Summarized data (mean±SE) showing gap closure (<i>a</i>) measured at 12 hr in Nor and Hyp PASMC treated with (BCS) or without (Control) BCS. **<i>P</i><0.01 vs. Control. The Cu-dependent cell motility (<i>b</i>) was determined by the percent changes in gap closure between Control and BCS-treated PASMC under Nor and Hyp conditions. *<i>P</i><0.05 vs. Nor. D: Summarized data (mean±SE) showing gap closure (<i>a</i>) measured at 12 hr in PASMC treated with (+CoCl₂) or without (-CoCl₂) CoCl₂ in the absence (Control) or presence (BCS) of BCS. *<i>P</i><0.05 vs. Control. The Cu-dependent cell motility (<i>b</i>) was determined by the percent changes in gap closure between Control and BCS-treated PASMC in the absence (-) or presence (+) of CoCl₂.</p