Jason X.-J. Yuan

Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip for Measuring Ion Channel Activity

The term “channelopathy” refers to a disease whose primary underlying mechanism(s) involve dysfunctional ion channel activity. Long QT syndrome and cystic fibrosis are two prime examples of channelopathies, where altered transport of K+, Na+, and/or Cl− ions, respectively, leads to pathogenesis. Such findings have aimed a new spotlight at the overall physiological relevance of transmembrane ion flux in disease development and became of critical importance to industry with discoveries that many drugs can unintentionally induce various channelopathies such as epileptic seizures (often from inhibition of neuronal human NaV1.1 Na+ channels), or torsade de pointes (a lethal long QT syndrome produced most often by inhibition of cardiac human ERG K+ channels). As discussed extensively elsewhere in this monograph, ion channels with selective permeability to Na+, K+, and Ca2+ ions modulate pulmonary vasoreactivity, remodeling, and vascular permeability. Although biochemical and molecular biology techniques have contributed, a significant proportion of our knowledge of the role of ion channels in pulmonary physiology has been garnered by measuring transmembrane ionic currents in intact cells using patch clamp electrophysiology. The purpose of this chapter is to introduce the basics of conventional and high-throughput patch clamp technology and how this can be used to advance the research into the cause of pulmonary vascular diseases.

Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle

Spanning the plasmalemmal membrane of all cells are many ion channels, exchangers, and transporters, all interacting to control vascular tone via the regulation of cytosolic Ca2+ concentration. Among these channels are voltage-gated K+, Na+, and Ca2+ channels, voltage-independent Ca2+ and nonselective cation channels, Cl− channels, and ligand-gated ion (K+, Ca2+, and Na+) channels. Plasmalemmal Ca2+–Mg2+ -ATPase (or Ca2+ pump) and Na+–K+ -ATPase (or Na+ pump) and an array of exchangers – the Na+–Ca2+ exchanger (NCX), Na+–H+ exchanger (NHE), and Na+–Mg2+ exchanger – all coexist to maintain ionic homeostasis in the cell. Comprehensive studies of ion channel function under both normal and pathophysiological conditions have been made possible owing to the development of cell isolation and electrophysiological techniques. This chapter introduces the ion channels and transporters present in the pulmonary vasculature (focusing on their expression and function) and discusses the potential pathogenic role of ion channels and transporters in the development of pulmonary hypertension.

Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature

Adult stem cells retain some capacity for self-renewal, although it is limited in comparison with embryonic stem cells, and are more restricted in their differentiation capacity. Adult stem cells are also commonly referred to as tissue specific stem cells. Some adult stem cells are still able to give rise to several specialized cell types (multipotent stem cells), whereas others are limited to a single specialized cell type (unipotent stem cells). Progenitor cells, the progeny of adult stem cells, differ from stem cells in that the potential for long-term self-renewal is lost. Scientifically, progenitor cells are more differentiated than stem cells. Primary stem cell progeny progenitor cells, usually known as multipotent adult progenitor cells, have full lineage-specific potential, whereas next-generation progenitors (oligopotent progenitors) are more lineage-restricted. This adult stem cell and progenitor cell hierarchy system exists to preserve a homeostatic repair and maintenance of the body, replenishing specialized cells and sustaining the routine cellular turnover in regenerative organs. Furthermore, the properties of these cells make them good candidates for targeted drug/gene delivery to specific organs; for example, mesenchymal stem cells (MSCs) have recently been shown to preferentially home to the lung. Adult stem and progenitor cells may be either circulating or resident in a particular tissue/organ system, including the lung and pulmonary vasculature. This chapter briefly describes the adult stem and progenitor cells currently identified in the pulmonary vasculature and introduces methodological approaches to successfully identify stem and progenitor cells.