Annual Review of Physiology - Volume 64, 2002

ATP-binding cassette (ABC) transporters located in the hepatocyte canalicular membrane of mammalian liver are critical players in bile formation and detoxification. Although ABC transporters have been well characterized functionally, only recently have several canalicular ABC transporters been cloned and their molecular nature revealed. Subsequently, development of specific antibodies has permitted a detailed investigation of ABC transporter intrahepatic distribution under varying physiological conditions. It is now apparent that there is a complex array of ABC transporters in hepatocytes. ABC transporter molecules reside in intrahepatic compartments and are delivered to the canalicular domain following increased physiological demand to secrete bile. Insufficient amounts of ABC transporters in the bile canalicular membrane result in cholestasis (i.e., bile secretory failure). Therefore, elucidation of the intrahepatic pathways and regulation of ABC transporters may help to understand the cause of cholestasis at a molecular level and provide clues for novel therapies.

Hepatocytes possess chloride channels at the plasma membrane and in multiple intracellular compartments. These channels are required for cell volume regulation and acidification of intracellular organelles. Evidence also supports a role of chloride channels in modulation of apoptosis and cell growth. Swelling- and Ca²⁺-activated chloride channels have been identified in hepatocyte plasma membranes, and chloride channels have been observed in the membranes of lysosomes, endosomes, Golgi, endoplasmic reticulum, mitochondria, and the nucleus. This review summarizes the functions of these channels and discusses the specific channel molecules they may represent. Chloride channel molecules shown to be expressed in hepatocytes include members of the ClC channel family (ClC-2, ClC-3, ClC-5, and ClC-7), members of the newly identified CLIC family of intracellular chloride channels (CLIC-1 and CLIC-4), the mitochondrial voltage-dependent anion channel, and a newly identified intracellular channel, MCLC (Mid-1 related chloride channel). Current understanding does not include a molecular identification of most of the observed channel functions, but details of the molecular properties of these channel molecules should allow future identification and further understanding of chloride channel function in hepatocytes.

Bile salts are the major organic solutes in bile and undergo extensive enterohepatic circulation. Hepatocellular bile salt uptake is mediated predominantly by the Na⁺-taurocholate cotransport proteins Ntcp (rodents) and NTCP (humans) and by the Na⁺-independent organic anion-transporting polypeptides Oatp1, Oatp2, and Oatp4 (rodents) and OATP-C (humans). After diffusion (bound by intracellular bile salt–binding proteins) to the canalicular membrane, monoanionic bile salts are secreted into bile canaliculi by the bile salt export pump Bsep (rodents) or BSEP (humans). Both belong to the ATP-binding cassette (ABC) transporter superfamily. Dianionic conjugated bile salts are secreted into bile by the multidrug-resistance-associated proteins Mrp2/MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by non-ionic diffusion and the apical sodium-dependent bile salt transporter Asbt/ASBT, transported back into the periductular capillary plexus by Mrp3/MRP3 [and/or a truncated form of Asbt (tAsbt)], and subjected to cholehepatic shunting. The major portion of biliary bile salts is aggregated into mixed micelles and transported into the intestine, where they are reabsorbed by apical Oatp3, the apical sodium-dependent bile salt transporter (ASBT), cytosolic intestinal bile acid-binding protein (IBABP), and basolateral Mrp3/MRP3 and tAsbt. Transcriptional and posttranscriptional regulation of these enterohepatic bile salt transporters is closely related to the regulation of lipid and cholesterol homeostasis. Furthermore, defective expression and function of bile salt transporters have been recognized as important causes for various cholestatic liver diseases.

Hereditary hemochromatosis (HH) is a common inborn error of iron metabolism characterized by excess dietary iron absorption and iron deposition in several tissues. Clinical consequences include hepatic failure, hepatocellular carcinoma, diabetes, cardiac failure, impotence, and arthritis. Despite the discovery of the mutation underlying most cases of HH, considerable uncertainty exists in the mechanism by which the normal gene product, HFE, regulates iron homeostasis. Knockout of the HFE gene clearly confers the HH phenotype on mice. However, studies on HFE expressed in cultured cells have not yet clarified the mechanism by which HFE mutations lead to increased dietary iron absorption. Recent discoveries suggest other genes, including a second transferrin receptor and the circulating peptide hepcidin, participate in a shared pathway with HFE in regulation of iron absorption. This review summarizes our current understanding of the relationship between iron stores and absorption and presents models to explain the dysregulated iron homeostasis in HH.

Lung cancer is the most common cause of cancer death in the United States, killing more than 156,000 people every year. In the past two decades, significant progress has been made in understanding the molecular and cellular pathogenesis of lung cancer. Abnormalities of proto-oncogenes, genetic and epigenetic changes of tumor suppressor genes, the role of angiogenesis in the multistage development of lung cancer, as well as detection of molecular abnormalities in preinvasive respiratory lesions, have recently come into focus. Efforts are ongoing to translate these findings into new clinical strategies for risk assessment, chemoprevention, early diagnosis, treatment selection, and prognosis and to provide new targets and methods of treatment for lung cancer patients. All these strategies should aid in reducing the number of newly diagnosed lung cancer cases and in increasing the survival and quality of life of patients with lung cancer.

Host defenses at the mucosal surface of the airways evolved to present many layers of protection against inhaled microbes. Normally, the intrapulmonary airways are sterile. Airway secretions contain numerous factors with antimicrobial activity that contribute to innate defenses. Many protein and peptide components exert bacteriostatic or bacteriocidal effects against a wide variety of organisms and may act in synergistic or additive combinations. The β-defensins are a relatively recently described family of peptide antimicrobials that are widely expressed at mucosal surfaces, including airway and submucosal gland epithelia. These small cationic peptides are products of individual genes that exhibit broad-spectrum activity against bacteria, fungi, and some enveloped viruses. Their expression in airway epithelia may be constitutive or inducible by bacterial products or pro-inflammatory cytokines. β-defensins also act as chemokines for adaptive immune cells, including immature dendritic cells and T cells via the CCR6 receptor, and provide a link between innate and adaptive immunity. Alterations in the function of the β-defensins may contribute to disease states. Here we review much of the biology of the β-defensins, including gene discovery, genomic organization, molecular structure, regulation of expression, and function.

Endothelial nitric oxide synthase (eNOS) is expressed in vascular endothelium, airway epithelium, and certain other cell types where it generates the key signaling molecule nitric oxide (NO). Diminished NO availability contributes to systemic and pulmonary hypertension, atherosclerosis, and airway dysfunction. Complex mechanisms underly the cell specificity of eNOS expression, and co- and post-translational processing leads to trafficking of the enzyme to plasma membrane caveolae. Within caveolae, eNOS is the downstream target member of a signaling complex in which it is functionally linked to both typical G protein-coupled receptors and less typical receptors such as estrogen receptor (ER) α and the high-density lipoprotein receptor SR-BI displaying novel actions. This compartmentalization facilitates dynamic protein-protein interactions and calcium- and phosphorylation-dependent signal transduction events that modify eNOS activity. Further understanding of these mechanisms will enable us to take preventive and therapeutic advantage of the powerful actions of NO in multiple cell types.

Recent studies in transgenic mice have revealed important insights into the roles of GM-CSF in regulation of surfactant homeostasis and lung host defense. Interruption of the GM-CSF signaling pathway by targeted ablation of the GM-CSF gene or its receptor (GM^−/− or GM Rβc^−/− mice, respectively) resulted in pulmonary alveolar proteinosis (PAP) but no hematologic abnormalities. Alveolar macrophages from GM^−/− mice have reduced capacity for surfactant catabolism, cell adhesion, phagocytosis, bacterial killing, Toll-receptor signaling, and expression of various pathogen-associated molecular pattern recognition receptors, suggesting arrest at an early stage of differentiation. PAP and abnormalities of alveolar macrophage function were corrected by local expression of GM-CSF in the lung, and expression of the transcription factor PU.1 in alveolar macrophages of GM^−/− mice rescued most defects. Recently, a strong association of auto-antibodies to GM-CSF or GM-CSF receptor gene mutations with PAP has implicated GM-CSF signaling abnormalities in the pathogenesis of PAP in humans. Together, these observations demonstrate that GM-CSF has a critical role in regulation of surfactant homeostasis and alveolar macrophage innate immune functions in the lung.

The diuretic-sensitive cotransport of cations with chloride is mediated by the cation-chloride cotransporters, a large gene family encompassing a total of seven Na-Cl, Na-K-2Cl, and K-Cl cotransporters, in addition to two related transporters of unknown function. The cation-chloride cotransporters perform a wide variety of physiological roles and differ dramatically in patterns of tissue expression and cellular localization. The renal-specific Na-Cl cotransporter (NCC) and Na-K-2Cl cotransporter (NKCC2) are involved in Gitelman and Bartter syndrome, respectively, autosomal recessive forms of metabolic alkalosis. The associated phenotypes due to loss-of-function mutations in NCC and NKCC2 are consistent, in part, with their functional roles in the distal convoluted tubule and thick ascending limb, respectively. Other cation-chloride cotransporters are positional candidates for Mendelian human disorders, and the K-Cl cotransporter KCC3, in particular, may be involved in degenerative peripheral neuropathies linked to chromosome 15q14. The characterization of mice with both spontaneous and targeted mutations of several cation-chloride cotransporters has also yielded significant insight into the physiological and pathophysiological roles of several members of the gene family. These studies implicate the Na-K-2Cl cotransporter NKCC1 in hearing, salivation, pain perception, spermatogenesis, and the control of extracellular fluid volume. Targeted deletion of the neuronal-specific K-Cl cotransporter KCC2 generates mice with a profound seizure disorder and confirms the central role of this transporter in modulating neuronal excitability. Finally, the comparison of human and murine phenotypes associated with loss-of-function mutations in cation-chloride cotransporters indicates important differences in physiology of the two species and provides an important opportunity for detailed physiological and morphological analysis of the tissues involved.

The recent knowledge of the renal epithelial transport systems has exploded with the identification, cloning, and characterization of a large number of membrane transport proteins. The fundamental aspects of these transporters are beginning to emerge at the molecular level and are summarized in the accompanying contributions in this volume of the Annual Review of Physiology. The aim of my review is to integrate this body of knowledge with the understanding of the clinical disorders of human mineral homeostasis that accompany gain, loss, or dysregulation of function of these transport systems. The specific focus is on the best defined human clinical syndromes in which there are derangements in K⁺ and Mg²⁺ homeostasis.

The epithelial sodium channel (ENaC) expressed in aldosterone-responsive epithelial cells of the kidney and colon plays a critical role in the control of sodium balance, blood volume, and blood pressure. In lung, ENaC has a distinct role in controlling the ionic composition of the air-liquid interface and thus the rate of mucociliary transport. Loss-of-function mutations in ENaC cause a severe salt-wasting syndrome in human pseudohypoaldosteronism type 1 (PHA-1). Gain-of-function mutations in ENaC β and γ subunits cause pseudoaldosteronism (Liddle's syndrome), a severe form of salt-sensitive hypertension. This review discusses genetically defined forms of a salt sensitivity and salt resistance in human monogenic diseases and in animal models mimicking PHA-1 or Liddle's syndrome. The complex interaction between genetic factors (ENaC mutations) and the risk factor (salt intake) can now be studied experimentally. The role of single-nucleotide polymorphisms (SNPs) in determining salt sensitivity or salt resistance in general populations is one of the main challenges of the post-genomic era.

Genetic disorders of acid-base transporters involve plasmalemmal and organellar transporters of H⁺, HCO3⁻, and Cl⁻. Autosomal-dominant and -recessive forms of distal renal tubular acidosis (dRTA) are caused by mutations in ion transporters of the acid-secreting Type A intercalated cell of the renal collecting duct. These include the AE1 Cl⁻/HCO3⁻ exchanger of the basolateral membrane and at least two subunits of the apical membrane vacuolar (v)H⁺-ATPase, the V1 subunit B1 (associated with deafness) and the V0 subunit a4. Recessive proximal RTA with ocular disease arises from mutations in the electrogenic Na⁺-bicarbonate cotransporter NBC1 of the proximal tubular cell basolateral membrane. Recessive mixed proximal-distal RTA accompanied by osteopetrosis and mental retardation is associated with mutations in cytoplasmic carbonic anhydrase II. The metabolic alkalosis of congenital chloride-losing diarrhea is caused by mutations in the DRA Cl⁻/HCO3⁻ exchanger of the ileocolonic apical membrane. Recessive osteopetrosis is caused by deficient osteoclast acid secretion across the ruffled border lacunar membrane, the result of mutations in the vH⁺-ATPase V0 subunit or in the CLC-7 Cl⁻ channel. X-linked nephrolithiasis and engineered deficiencies in some other CLC Cl⁻ channels are thought to represent defects of organellar acidification. Study of acid-base transport disease-associated mutations should enhance our understanding of protein structure-function relationships and their impact on the physiology of cell, tissue, and organism.