Adriana M. Zimnicka

Human Copper Transporter hCTR1 Mediates Basolateral Uptake of Copper into Enterocytes IMPLICATIONS FOR COPPER HOMEOSTASIS

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.

Enhanced Ca 2+ -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 mmol/L 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.

Copper entry into human cells: progress and unanswered questions

In this brief review we summarize what is known about the role of hCTR1 in mediating the entry of copper into human cells. There is a body of information that clearly identifies this protein as being a major source (though not the only source) of copper entry into human cells, and thus a crucial element of copper homeostasis. However, much remains that is poorly understood and key aspects of the physiological roles of hCTR1 and its regulation are only superficially appreciated. The particular characteristics of a transport process that in vivo involves the binding, transmembrane transport and release of a substrate that is not present in a free form in the intracellular or extracellular compartments poses particular challenges that are not encountered in the transport of more familiar physiologically important metal cations. Thus much of what we have learned about the more commonly encountered transported ions provides an inadequate model for studies of copper homeostasis. In this article we review progress made and identify the major questions that need to be resolved before an adequate description is attained of how copper entry into human cells is mediated and regulated by hCTR1.

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.

Chronic hypoxia selectively enhances L- and T-type voltage-dependent Ca2+ channel activity in pulmonary artery by upregulating Cav1.2 and Cav3.2

Hypoxia-induced pulmonary hypertension (HPH) is characterized by sustained pulmonary vasoconstriction and vascular remodeling, both of which are mediated by pulmonary artery smooth muscle cell (PASMC) contraction and proliferation, respectively. An increase in cytosolic Ca²⁺ concentration ([Ca²⁺]cyt) is a major trigger for pulmonary vasoconstriction and an important stimulus for cell proliferation in PASMCs. Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) is an important pathway for the regulation of [Ca²⁺]cyt. The potential role for L- and T-type VDCC in the development of HPH is still unclear. Using a hypoxic-induced pulmonary hypertension mouse model, we undertook this study to identify if VDCC in pulmonary artery (PA) are functionally upregulated and determine which type of VDCC are altered in HPH. Mice subjected to chronic hypoxia developed pulmonary hypertension within 4 wk, and high-K⁺- and U-46619-induced contraction of PA was greater in chronic hypoxic mice than that in normoxic control mice. Additionally, we demonstrate that high-K⁺- and U-46619-induced Ca²⁺ influx in PASMC is significantly increased in the hypoxic group. The VDCC activator, Bay K8864, induced greater contraction of the PA of hypoxic mice than in that of normoxic mice in isometric force measurements. L-type and T-type VDCC blockers significantly attenuated absolute contraction of the PA in hypoxic mice. Chronic hypoxia did not increase high-K⁺- and U-46619-induced contraction of mesenteric artery (MA). Compared with MA, PA displayed higher expression of calcium channel voltage-dependent L-type α1C-subunit (Cav1.2) and T-type α1H-subunit (Cav3.2) upon exposure to chronic hypoxia. In conclusion, both L-type and T-type VDCC were functionally upregulated in PA, but not MA, in HPH mice, which could result from selectively increased expression of Cav1.2 and Cav3.2.

Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

Src-induced phosphorylation of Cav-1 is analyzed using live TIRF and FRET microscopy, as well as by biochemical analysis. Cav1 phosphorylation destabilizes plasma membrane–associated Cav-1 oligomers and thereby is crucial for regulating the fission of caveolae from the plasma membrane in vascular endothelial cells.

Dihydropyridine Ca2+ Channel Blockers Increase Cytosolic [Ca2+] by Activating Ca2+-sensing Receptors in Pulmonary Arterial Smooth Muscle Cells

Rationale: An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca2+ channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH). Objective: Our previous study demonstrated that the Ca2+-sensing receptor (CaSR) was upregulated and the extracellular Ca2+-induced increase in [Ca2+]cyt was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca2+]cyt by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC. Methods and Results: The nifedipine-mediated increase in [Ca2+]cyt in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca2+]cyt, whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca2+]cyt. Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca2+]cyt in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca2+]cyt. Conclusions: The dihydropyridine derivatives increase [Ca2+]cyt by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca2+ channels; therefore, it is possible that the use of dihydropyridine Ca2+ channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary 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.

Inhibition of the Ca2+-sensing receptor rescues pulmonary hypertension in rats and mice

A recent study from our group demonstrated that the Ca2+-sensing receptor (CaSR) was upregulated, and the extracellular Ca2+-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt) was enhanced in pulmonary arterial smooth muscle cells from patients with idiopathic pulmonary arterial hypertension and animals with experimental pulmonary hypertension (PH). However, it is unclear whether CaSR antagonists (for example, NPS2143) rescue the development of experimental PH. We tested the rescue effects of NPS2143 in rats with monocrotaline (MCT)-induced PH and mice with chronic hypoxia-induced PH. For the NPS2143 treatment group, rats and mice were i.p. injected with NPS2143 once per day from days 14 to 24. Four weeks after MCT injection or exposure to normobaric hypoxia, the right ventricular (RV) systolic pressure, right heart hypertrophy (RV/LV+S ratio) and RV myocardial fibrosis were rescued or nearly restored to normal levels by NPS2143 treatment. The rescue effects of NPS2143 on experimental PH further support a critical role for the CaSR in the PH mechanism. Therefore, NPS2143 may be a promising potential treatment for pulmonary arterial hypertension.

Functional characterization of voltage-dependent Ca2+ channels in mouse pulmonary arterial smooth muscle cells: divergent effect of ROS

Electromechanical coupling via membrane depolarization-mediated activation of voltage-dependent Ca(2+) channels (VDCC) is an important mechanism in regulating pulmonary vascular tone, while mouse is an animal model often used to study pathogenic mechanisms of pulmonary vascular disease. The function of VDCC in mouse pulmonary artery (PA) smooth muscle cells (PASMC), however, has not been characterized, and their functional role in reactive oxygen species (ROS)-mediated regulation of vascular function remains unclear. In this study, we characterized the electrophysiological and pharmacological properties of VDCC in PASMC and the divergent effects of ROS produced by xanthine oxidase (XO) and hypoxanthine (HX) on VDCC in PA and mesenteric artery (MA). Our data show that removal of extracellular Ca(2+) or application of nifedipine, a dihydropyridine VDCC blocker, both significantly inhibited 80 mM K(+)-mediated PA contraction. In freshly dissociated PASMC, the maximum inward Ca(2+) currents were -2.6 ± 0.2 pA/pF at +10 mV (with a holding potential of -70 mV). Window currents were between -40 and +10 mV with a peak at -15.4 mV. Nifedipine inhibited currents with an IC(50) of 0.023 μM, and 1 μM Bay K8644, a dihydropyridine VDCC agonist, increased the inward currents by 61%. XO/HX attenuated 60 mM K(+)-mediated increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) influx through VDCC in PASMC. Exposure to XO/HX caused relaxation in PA preconstricted by 80 mM K(+) but not in aorta and MA. In contrast, H(2)O(2) inhibited high K(+)-mediated increase in [Ca(2+)](cyt) and caused relaxation in both PA and MA. Indeed, RT-PCR and Western blot analysis revealed significantly lower expression of Ca(V)1.3 in MA compared with PA. Thus our study characterized the properties of VDCC and demonstrates that ROS differentially regulate vascular contraction by regulating VDCC in PA and systemic arteries.