ATP regulation of epithelial Cl⁻ channels - new challenges?

ATP is released from many cells into neuronal synapses, interstitium and body cavities where it acts as a co-transmitter or as an autocrine or paracrine regulator before being broken down by various ecto-nucleotidases. ATP and its metabolites affect many cellular functions, including epithelial transport in, for example, exocrine glands. By the 1980s it was already recognized that ATP had potent neurotransmitter-like effects on salivary glands, but ATP regulation of salivary secretion remains unclear. Firstly, we do not know where ATP is released. It could be released from the nerves that innervate the glands or directly from acinar vesicles or secretory granules into the lumen following muscarinic stimulation, as in pancreatic acini (Novak, 2003). The regulation of release is also not known - possibly it involves neural impulses, osmotic swelling or mechanical stress. Mechanical stress especially could be an important stimulus since the salivary glands are exposed to contraction of myoepithelial cells, surrounding secretory endpieces of some glands, and of the jaw muscles during chewing and speech.

ATP and other nucleotides interact with purinergic P2 receptors on the plasma membrane. Salivary glands have numerous P2 receptors belonging to the P2X family of ligand-gated cation channels and to the P2Y family of seven transmembrane-spanning receptors, which are coupled to G-proteins. In native salivary acini P2X₇ and P2X₄ receptors are functionally predominant, while P2Y₁ receptors are expressed in immature glands. P2Y₂ receptors are also present but are up-regulated in response to tissue damage or cell culture (Ahn et al. 2000). Many effects of ATP on salivary acini have been reported during the last 20 years. ATP induces transient outward currents due to activation of K⁺ channels, and inward Na⁺ (Ca²⁺) currents through activation of P2X receptors. ATP also stimulates Na⁺-H⁺ exchange, Na⁺-K⁺-2Cl⁻ cotransport, cell volume changes, amylase release and, at high concentrations, non-selective permeability (McMillian et al. 1988; Novak, 2003). Most of these actions could promote secretion; however the key event for secretion is the opening of the luminal Cl⁻ channels. Whether ATP affects Cl⁻ channels under physiological conditions has not yet been resolved. ATP-induced Cl⁻ currents were not detected in salivary acini until the study by Zeng et al. (1997). Similarly, ATP/UTP-stimulated Cl⁻ currents are detected in secretory pancreatic ducts only when cultured cells are used, and have been most often studied using cells with the cystic fibrosis (CF) defect (Novak, 2003). The issue of ATP stimulation of Cl⁻ channels is relevant to CF, as the defective CFTR-Cl⁻ channel could be bypassed by stimulation of Ca²⁺-regulated Cl⁻ channels. In salivary glands there is a palette of Cl⁻ channels that includes Ca²⁺-regulated Cl⁻ channels, inwardly rectifying ClC-2 and possibly ClC-3b channels, volume-activated Cl⁻ channels, and CFTR-Cl⁻ channels. The most important channel type during normal (i.e. acetylcholine-induced) secretion seems to be the Ca²⁺-regulated Cl⁻ channel (Begenisich & Melvin, 1998). However, ATP increases both intracellular Ca²⁺ and diacylglycerol, therefore Ca²⁺-regulated Cl⁻ channels and (Ca²⁺-independent) protein kinase C-stimulated CFTR-Cl⁻ channels are both good candidates (Novak, 2003).

In this issue of The Journal of Physiology, Arreola & Melvin (2003) readdress the question of ATP-stimulated Cl⁻ channels in mouse parotid acini. The outcome is quite unexpected, both with respect to the ATP receptor and the effector, i.e. Cl⁻ current (I_ATPCl). The authors demonstrate ATP stimulation of whole-cell Cl⁻ currents by extracellular ATP. Since similar currents are seen using cells from mice with targeted disruption of known Cl⁻ channel genes coding for ClC-2, ClC-3 and CFTR, none of these channels could mediate I_ATPCl. They study I_ATPCl in more detail at positive clamp voltages (as it decays rapidly in the negative range), and find it to be independent of intracellular Ca²⁺, slightly outwardly rectifying, resistant to the usual Cl⁻ channel inhibitors and with an unusual anion selectivity sequence. Thus, it does not correspond to any known channels, raising the possibility that we are dealing with a new Cl⁻ channel type. Some properties of the ATP-activated chloride currents resemble those of single channels that have been recorded previously on the luminal membrane of pancreatic acinar cells, but the effects of ATP were not assessed in those earlier studies (Zdebik et al. 1997). We are just beginning to understand the complexity of Cl⁻ channels and their functions, and many channels are not yet identified at the molecular level. For salivary acinar physiology, we will have to see how these proposed ATP-stimulated Cl⁻ channels behave with normal cellular Ca²⁺ concentrations, and whether they can be activated at physiological membrane potentials and temperatures. Moreover, in order to elicit salivary secretion, sustained Cl⁻ channel activity will have to be matched by K⁺ channels to keep the driving force, and by continuous action of other ion transporters.

Another unexpected twist in the new work relates to purinergic receptors. There are a number of P2 receptor inhibitors available, which have been useful in studies of other cells. Arreola & Melvin (2003) show that Brilliant Blue G, PPADS and Suramin do not affect I_ATPCl. Since antagonists of G proteins and purinoceptors had no effect, the results imply that P2Y receptors are not involved in the activation of Cl⁻ channels by ATP. However, Cibacron Blue 3GA and DIDS were effective in inhibiting the Na⁺ current, suggesting that P2X receptors were nevertheless stimulated. Thus, there is a divorce between the Na⁺ channel function of P2X receptors and Cl⁻ channel activation, raising the possibility that ATP regulates Cl⁻ channels directly, perhaps serving as a substrate as it does for ecto-protein kinases and ecto-nucleotidases at the cell surface. Alternatively, there may be a P2X receptor with blocked cation pores that can still regulate separate anion channels or a novel P2 receptor that is permeable to Cl⁻ and not sensitive to known inhibitors under the conditions used in the study.

This new study by Arreola & Melvin (2003) suggests intriguing new avenues that should be explored further to fully understand how ATP regulates Cl⁻ channels and salivary secretion. Many challenges lie ahead, most urgently the molecular identification of ATP receptors and ATP-stimulated Cl⁻ channels and their localization in acini with respect to the site of ATP release in the gland.