Airway nerves and protein phosphatases

General Pharmacology 32 (1999) 287–298

Review article

Airway nerves and protein phosphatases S. Harrison

a,b,

*, C.P. Page a, D. Spina

a

a Department of Respiratory Medicine and Allergy, GKT School of Medicine, King’s College London, London SE5 9PJ, UK Division of Pharmacology and Therapeutics, GKT School of Biomedical Science, King’s College London, London SW3 6LX, UK Manuscript received June 2, 1998; accepted manuscript June 15, 1998

b

Abstract Much attention has focused on the important role played by phosphatases in the control of gene transcription, cell differentiation and memory regulation. It is also clear that phosphatases may regulate a number of biochemical pathways which can modulate cellular function. Of particular interest is the role of phosphatases in the control of neuronal function. Alterations in neuronal function may contributed to the heightened airways responsiveness observed in asthma to a number of physiological stimuli including distilled water, sulfur dioxide, metabisulfite, hypertonic saline, exercise, allergens, viruses and cold air. An understanding of the mechanisms which regulate the function of sensory nerves could have important clinical implications. In this review we will highlight a number of studies that have investigated the role of phosphatases in the regulation of airway nerve function.  1999 Elsevier Science Inc. All rights reserved. Keywords: Airway smooth muscle; Protein phosphatases; Sensory nerves

Asthma is characterized by cough and bronchospasm which can be elicited by substances which would otherwise have minimal activity in the airways of healthy individuals. Bronchial hyperresponsiveness is a term used to define such exaggerated responses to allergic and nonallergic stimuli in asthmatics. A number of mechanisms have been proposed to account for this phenomenon including hypertrophied airway smooth muscle, facilitated excitation of adjacent myocytes, loss of homeostatic bronchodilator mechanisms, loss of epithelial integrity, and reduction of luminal diameter secondary to increased volume of airway smooth muscle and mucosal edema (Sterk and Bel, 1989). It has also been suggested that an alteration in the function of sensory nerves might lead to a dramatic increase in responsiveness to stimuli well after the offending insult has been removed. It has therefore been suggested that bronchial hyperresponsiveness may be likened to hyperalgesia in the skin, with sensory nerves principally involved in this response (Adcock and Garland, 1993). Thus, many investigators in this field have sought to understand the function of airway nerves as they may offer novel targets for the development of new anti-inflammatory drugs. In this regard there is a paucity of data * Corresponding author. Tel.: 44 0171 333 4741; Fax: 44 0171 376 8150.

concerning the role of phosphatases in the control of neuronal function in the airways. A variety of cellular functions including gen transcription, cell differentiation, contractility, neurotransmission, and memory regulation involve phosphorylation of proteins (Cohen, 1989; Coghlan et al., 1995) which is dependent upon the relative activity of protein kinases and phosphatases (Cohen, 1988). Given the importance of protein phosphorylation in the context of cell function, abnormal protein phosphatase activity has been implicated in diseases such as cancer (Cantley et al., 1991), diabetes (Taylor et al., 1992), and inflammation (Parnetti et al., 1997). This review will give a brief discussion of the different classes of phosphatases and their role in the control of neuronal function with particular emphasis on nerves found in airways. 1. Classification of protein phosphatases Protein phosphatases are classified according to their substrate specificity, dependence upon metal ions for activity, and sensitivity to inhibitory or activator agents (Ingebritsen and Cohen, 1983; Cohen, 1985) of which there are four major classes. Type 1 protein phosphatase (PP1) dephosphorylates the b subunit of phosphorylase kinase which is inhibited by thermostable protein

0306-3623/99/$–see front matter  1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00204-3

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inhibitor-1 and inhibitor-2 (Cohen, 1989). The type 2 phosphatase (PP2) preferentially dephosphorylates the a subunit of phosphorylase kinase, is unaffected by the thermostable protein inhibitors (Ingebritsen and Cohen, 1983), and is further divided into PP2A, PP2B (calcineurin), and PP2C (Ingebritsen and Cohen, 1983; Brautigan et al., 1986). PP2A has no requirement for divalent metal ions for its activity, while PP2B is dependent upon calcium (Honkanen et al., 1991) and PP2C is Mg21-dependent and okadaic acid-insensitive (Ingebritsen and Cohen, 1983; Cohen, 1989; Cohen, 1997). More distantly related are the protein tyrosine phosphatases (PTP) of which over 40 have been characterized. They possess 230 amino acid catalytic domain and a number of regulatory subunits that are essential for subcellular localization and enzymatic activity (Mauro and Dixon, 1994). Finally, there are a group of novel protein phosphatases which are more closely related to PP1 and PP2 and include PP4, PP5, and PP6. 1.1. Protein phosphatase I Human PP1 has a molecular mass of 37 kDa (Tung and Cohen, 1984) and can be divided into three isoforms which demonstrate 85% homogeneity with each other (Favre et al., 1997). The diversity observed is due to the catalytic region of which there are four isoforms, a, g1, g2, and d (Sasaki et al., 1990) inhibited by heat stable proteins inhibitor-1 and inhibitor-2 (Depaoli Roach et al., 1994; Wera and Hemmings, 1995). Protein sequence variations among these isoforms are mainly confined to the carboxyl terminus (Sasaki et al., 1990), thought to play a regulatory role in the catalytic activity such as proteolysis (Martin et al., 1991) and phosphorylation (Yamano et al., 1994; Eto et al., 1995). The catalytic region interacts with a number of regulatory subunits (Cohen, 1988; Ohkura et al., 1989; Cohen, 1997) that contain a common motif (R/K) (V/I) xF (Johnson et al., 1996; Egloff et al., 1997). Regulatory subunits also modulate the substrate specificity of PP1 thus allowing the activity to be modulated by extracellular signals such as hormones and growth factors (Hubbard and Cohen, 1993). Genetic, pharmacological, and biochemical data show that PP1 modulates glycogen metabolism (Cohen, 1982), mitosis (Sakurada et al., 1992), meiosis (Huchon et al., 1981), muscle contraction (Shenolikar, 1994; Wera and Hemmings, 1995), and other cell-cycle events (Cyert and Thorner, 1989). In skeletal muscle, almost all of the active PP1 is associated with glycogen particles, myofibrils, and the sarcoplasmic reticulum (Cohen et al., 1989). The glycogen-associated PP1 (PP1G) is a heterodimer composed of a catalytic subunit associated with a 161 kDa glycogen-binding subunit which is responsible for the association with glycogen (Stralfors et al., 1985; Hiraga et al., 1987; Hubbard and Cohen, 1989). PP1G is 5–10 fold more active than the free cata-

lytic subunit in dephosphorylating the enzyme for glycogenolysis and glycogen synthesis (Ingebritsen and Cohen, 1983). In myofibrils of skeletal and cardiac muscle, PP1 is distinct from that of PP1G, as it has enhanced activity towards myosin and may be composed of the catalytic subunit of PP1 complex to other proteins responsible for the activity towards myosin or interaction with myofibrils (Chisholm and Cohen, 1988). 1.2. Protein phosphatase 2A PP2A exhibits sequence homology with PP1 (Berndt et al., 1987) and consists of two isoforms of which the catalytic subunits undergo reversible methylesterification at the carboxyl terminus (Lee et al., 1996) resulting in the alteration of the regulatory subunits (65 kDa) (de la Cruz et al., 1987; Stone et al., 1987; Arino et al., 1988; Favre et al., 1997). PP2A is a heterotrimeric complex consisting of a catalytic subunit (36–38 kDa), a structural subunit (60-65 kDa), and B subunit (54-74 kDa) (Cohen, 1989; Shenolikar and Nairn, 1991; Mumby and Walter, 1993). Cloning has identified two isoforms (a and b) for the structural subunit (Walter et al., 1989; Hemming et al., 1990) and catalytic subunit (Green et al., 1987; Stone et al., 1987). Messenger RNA (mRNA) encoding PP2Ab isoforms were 10-fold less abundant than those encoding PP2Aa isoforms (Khew Goodall and Hemmings, 1988). It has been demonstrated that there is specific binding of Ba and Bb subunits to the microtubule-associated protein tau, and this is thought to be involved in a number of cellular processes such as metabolic regulation and cell signalling (Sontag et al., 1996). The B subunit seems to suppress activity towards some substrates, but enhances dephosphorylation of other substrates (Cohen, 1997). There are suggestions that the B subunit may participate in the regulation of activity as observed for inhibitor-1 and inhibitor-2 (Cohen et al., 1989; Shenoliker and Nairn, 1991; Bollen and Stalmans, 1992). PP2A catalytic subunit is phosphorylated by tyrosine kinases on Tyr307, two residues upstream of its carboxyl terminus (Chen et al., 1994). PP2A is thought to regulate events during the onset of mitosis (Pfaller et al., 1991) and the inhibition of endocytic vesicle fusion (Woodman et al., 1992). PP2A has been shown to be present in fibroblast nuclei (Turowski et al., 1995), microtubules (Sontag et al., 1995), and neurofilaments (Saito et al., 1995). Both PP1 and PP2A have been linked to the regulation of calcium/calmodulin-dependent protein kinase. Autophosphorylation of Thr305-306 located within the calmodulin binding domain is closely correlated with the loss of sensitivity to calmodulin and reversed by PP1 or PP2A (Patton et al., 1990). A role for PP1/PP2A in mitosis has been supported by recent findings that mitotic arrest in human leukemia cell lines occurs when incu-

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bated with the PP1/PP2A inhibitor okadaic acid (Zheng et al., 1991; Sakurada et al., 1992). 1.3. Protein phosphatase 2B (calcineurin) PP2B, also known as calcineurin, is a Ca21-dependent calmodulin stimulated enzyme regulated by Ca21 (19 kDa Ca21-binding subunit) in the absence of calmodulin (Cohen et al., 1989; Yakel, 1997). PP2B is a heterodimer composed of a catalytic subunit (60 kDa) and a myristoylated regulatory B subunit (19 kDa) (Klee et al., 1988). The catalytic domain is located at residues 14342, B-binding domain in residues 343-373, and the calmodulin binding domain in position 390-414 (Griffith et al., 1995). The B subunit is structurally related to calmodulin (17 kDa, 30–50% homology), containing four EF-hand Ca21 binding loops (Guerini et al., 1989; Guerini, 1997). The B subunit and calmodulin are both needed for calcineurin to be fully activated; they both bind to the calcineurin catalytic subunit without any cross-binding. The B subunit is highly associated with the catalytic subunit independently of Ca21; phosphatase activity only occurs when Ca21 binds to the B subunit. It has been proposed that the region between calcineurin B (343-373) and the calmodulin binding domain (374-389) bends back to bring the auto-inhibitory domain in contact with the active site thereby inhibiting phosphatase activity (Griffith et al., 1995). Subsequent activation of calcineurin involves the binding of Ca21/calmodulin complex which disrupts the calmodulin binding domain and the calcineurin B binding helix, inducing a conformational change which displaces the auto-inhibitory domain from the catalytic domain (Klee et al., 1988). PP2B is one of the major calmodulin binding proteins in the brain comprising about 1% of total protein content. PP2B has been found in high density in granule cells of the cerebellum, caudate putamen, neurostriatum, and hippocampus, and is thought to be responsible for N-methyl-D-aspartate (NMDA) receptor signalling (Klee et al., 1988; Shenolikar and Nairn, 1991; Wera and Hemmings, 1995). PP2B is found in cell bodies, postsynaptic densities, dendrites, axons, spine, and presynaptic terminals (Klee et al., 1988). In rat cortical neurones primary cultures, NMDA receptor-induced neurotoxicity is thought to be mediated by nitric oxide (NO) and blocked by inhibiting PP2B (Dawson et al., 1993). Nitric oxide synthase (NOS) is a substrate for PP2B and it has been proposed that dephosphorylation of NOS enhances its catalytic activity, resulting in NO formation and neurotoxicity (Dawson et al., 1993). Neuronal PP2B is thought to modulate l-type Ca21 channels and voltage-gated K1 channels and down-regulate tyrosine phosphatase activity of CD45 (Eckert and Chad, 1984; Hosey et al., 1986; Hoger et al., 1991; Ostergaard and Trowbridge, 1991; Imredy and Yue, 1994). In molluscan neurones, PP2B increases the rate

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of inactivation of the voltage-gated Ca21 current (Chad and Eckert, 1986), and it is thought that Ca21 influx through the Ca21 channels during their voltage-dependent opening activates PP2B, which in turn dephosphorylates the channels, leading to inactivation of the Ca21 currents. Similarly, PP2A and PP2B dephosphorylate sites involved in phosphorylation of Na1 channels in rat brain (Chen et al., 1995). The regulation of Ca21 entry through ion channels by PP2B is thought to be important in hippocampal long-term depression (Mulkey et al., 1994), migration of neutrophils (Hendey et al., 1992), and motility and outgrowth of growth cones (Ferreira et al., 1993; Chang et al., 1995). In T lymphocytes, PP2B is activated by a rise in cytoplasmic Ca21, causing dephosphorylation of the cytoplasmic form of transcription factor nuclear factor of activated T cells (NFAT) which initiates transcription of T-cell growth factor IL-2 (Jain et al., 1993; Perrino et al., 1995). 1.4. Protein phosphatase 2C PP2C (also known as PP1A) is a monomeric protein (42-45 kDa) (Ingebritsen and Cohen, 1983) which consists of two isoforms, PP2C1 and PP2C2, based on amino acid sequence in rabbit skeletal muscle and liver (McGowan et al., 1987). PP2C (42-48 kDa) requires Mg21 for activity and preferentially dephosphorylates the a subunit of phosphorylase 6 kinase (Hiraga et al., 1981; Tsuiki et al., 1988). PP2C activity has been reported in the brain and this is greater than the activity reported in skeletal muscle and liver (Ingebritsen and Cohen, 1983). In situ hybridization of mRNA for PP2C revealed localization to granule cells and Purkinje cells of the cerebellum, hippocampus, and plexus choroideus of the lateral ventricles, demonstrating a wide distribution throughout the brain (Abe et al., 1992). Phosphorylation in the cerebellar granule cells is okadaic acidinsensitive and consists of Mg21-dependent activity, which supports the view that PP2C is present in these cells (Fukunaga et al., 1993). PP2C also contributes to dephosphorylation of synaptic junction proteins such as calmodulin kinase II (Shields et al., 1984). 1.5. Protein tyrosine phosphatase Protein tyrosine phosphatases (PTPs) are a structurally diverse family, comprised of receptors with the ability to transmit signals directly across the membrane and cytoplasmic enzymes which act both positively and negatively in the control of cell function (Fischer et al., 1991; Walton and Dixon, 1993). PTPs show no sequence homology with serine/threonine phosphatases or with the broad-specificity phosphatases, but share a structural relationship with the surface leukocyte common antigen CD45 (Charbonneau et al., 1988). PTPs can be divided into 4 groups: tyrosine specific phosphatases, VH-1-like dual specificity phosphatases, cdc25 (cell-division control) and low molecular weight

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(LMW) phosphatases. They all share a common motif (Cx5R) which forms a distinct cradle and phosphate binding loop in which oxyanions bind. Binding of the oxyanions result in a conformational change, swinging the conserved aspartic acid into the active site (Schubert et al., 1995). Unlike the other protein phosphatases, these do not require metal ions for catalysis (Fauman and Saper, 1996). The tyrosine-specific phosphatases are further divided into transmembrane receptor-like phosphatases and nonreceptor cytoplasmic phosphatases. The receptor-like PTPs have variable extracellular domains, a single transmembrane segment and one or two intracellular catalytic domains (Fashena and Zinn, 1995). When two catalytic domains are present, the carboxyl-terminal domain is usually catalytically inactive (Krueger and Saito, 1992). It is presumed that the binding of ligands may modulate phosphatase activity and/ or re-localize receptor tyrosine phosphatase thus making them available for potential substrates. There are a number of subtypes: R-PTPk and m are identical and thought to undergo homophilic cell-cell interaction and induce cell growth (Brady-Kalnay and Tonk, 1994; Sontag et al., 1995); R-PTPm is associated with a protein complex which contains cadherins and a- and b-catenins and is thought to be involved in cell adhesion (Brady-Kalnay and Tonk, 1994); R-PTPb is widely expressed in glial cells in the embryonic nervous system, and in the adult brain a form exists which lacks the transmembrane and catalytic domain, and is thought to interact with neural cell adhesion molecules (Barnea et al., 1994; Milev et al., 1994; Peles et al., 1995); R-PTPz contains an extracellular carbonic anhydrase domain that specifically binds a neural cell surface molecule, and may play a role in neuronal migration (Peles et al., 1995). The nonreceptor-like PTP has an extra catalytic segment which is involved in regulating catalytic activity through cellular localization (Mauro and Dixon, 1994). PTP1B and T-cell PTP contain carboxy-terminal hydrophobic segment that targets the catalytic domain in endoplasmic reticulum (Cool et al., 1990; Frangioni et al., 1992). The vaccinia virus late H1 gene, VH-1-like dual specificity phosphatases, has a human counterpart known as VHR (Fauman and Saper, 1996); these are thought to be responsible for activation of cyclin-dependent kinase-cyclin complexes during cell cycle (Aroca et al., 1995). VHR share a 5-stranded mixed b-sheet and 6 a-helices, with a phosphate binding loop and nearby acid loop (Denu and Dixon, 1995). VHR hydrolyses phosphotyrosine 40 times faster than phosphothreonine and 500 times faster than phosphoserine (Zhang et al., 1995). Cdc25 activates and initiates M phase by phosphorylating a wide range of cellular proteins (Fauman and Saper, 1996). Three isoforms for cdc25 have been isolated as A, B and C, and each isoform is responsible

for a distinct cyclin-dependent kinase-cyclin complex, Cdc25 enzymes dephosphorylate both Tyr15 and Thr14 of cyclin-dependent kinases (Sebastian et al., 1993). Low molecular weight PTPs (also known as acid phosphatase) are composed of only a catalytic domain. The phosphate-binding loop precedes the general acid loop in LMW PTPs (Fauman and Saper, 1996). No biological function for these protein phosphatases has yet been found, although LMW PTPs in vitro are specific for aryl phosphatases, for example, phosphotyrosine (Fauman and Saper, 1996). Other than the Cx5R motif, there is no similarity between the LMW phosphatases and other PTPs. 1.6. Novel protein phosphatase (PP4, PP5, and PP6) Apart from the most commonly known protein phosphatases (described above), there are also a group of novel protein phosphatases, referred to as PP4, PP5, and PP6. PP4 is thought to belong to PP1 and PP2A subfamilies, as they demonstrate about 60% homology with this group. Its localization is fairly widespread, found in centrosomes, nuclei, and cytoplasm suggesting that PP4 has a multitude of functions (da Cruz et al., 1988; Brewis et al., 1993). PP4 may be involved in centrosome duplication as it has been detected at various stages of mitosis (except telophase) (Brewis et al., 1993). PP5 has been found in all human tissues examined, suggesting that it is probably localized in the nuclei (Fischer et al., 1989). PP5 gene harbors a tetratricopeptide repeat (TPR) domain which is also found in proteins involved in regulation of RNA synthesis and mitosis (Boguski et al., 1990; Sikorksi et al., 1991; Chen et al., 1994). The TPR domain of PP5 is thought to be important for protein-protein interactions and subcellular targeting (Lamb et al., 1995). a-Amaritin (an inhibitor of RNA polymerase) causes an increase in levels of PP5 in the nucleolus, thus suggesting its involvement in the regulation of ribosomal RNA transcription (Cohen et al., 1996). Human PP5 is thought to be involved in cell growth due to the fact that high levels of PP5 are present in cells in logarithmic growth, compared to dividing or serum-deprived cells (Cohen et al., 1996). The four TPRs in PP5 are thought to bind to heat shock protein (hsp) 90 (Chen et al., 1996); both FKBP (FK binding protein) and cyclophilin also bind to hsp 90 via the TPR domain. Competition binding experiments with PP5 TPR domain suggest that these three proteins occupy a common binding site on hsp 90 (Silverstein et al., 1997). PP5 associates with progesterone receptor heterocomplexes (Picard et al., 1990) and glucocorticosteroids (Chen et al., 1994), suggesting that PP5 may involved in steroid receptor signalling. Human PP6, a 35 kDa protein which exhibits homology with PP2A (57%) and PP4 (59%), is also homologous to S. cerevisiae SIT4 (Mann et al., 1993; Bastians

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and Ponstingl, 1996). In S. cerevisiae, SIT4 is required for the passage from G1 to S phase in the cell cycle, thus allowing accumulation of G1 cyclin mRNA (Shimanuki et al., 1993; Bastians and Ponstingl, 1996). Thus, mammalian PP6 may play a role in regulating transcription. In human cells, three forms of PP6 mRNA are expressed in testis, heart, and skeletal muscle (Bastians and Ponstingl, 1996). 2. Protein phosphatase inhibitors The recent discovery of protein phosphatase inhibitors has proved invaluable in elucidating the function of protein phosphatases. Protein phosphatase inhibitors can be generally classed into three groups: PP1, PP2A and PP2B, and PTPs. 2.1. Protein phosphosphatase 1 & 2 A inhibitors PP1 and PP2A are sensitive to a number of natural products: calyculin A (Ishihara et al., 1989a), tautomycin (Cheng et al., 1987; MacKintosh and Klumpp, 1990), nodularins (Honkanen et al., 1991; An and Carmichael, 1994), motuporin (Craig et al., 1996), microcystins (Carmichael et al., 1988; Honkanen et al., 1990; An and Carmichael, 1994), and okadaic acid (Takai and Mieskes, 1991). Structurally, calyculin A is markedly different from okadaic acid, in fact, its profile for protein phosphatase inhibition differs greatly. Both compounds inhibit protein serine/threonine phosphatases; however, calyculin A (Ki for PP1 is 1 nM while the Ki for PP2A is 0.12 nM) is more potent than okadaic acid (Ki for PP1 is 147 nM, for PP2A 0.032 nM) at inhibiting PP1 while the reverse is true for PP2A (Bialojan and Takai, 1988; Ishihara et al., 1989b; Takai et al., 1995); each only partially inhibits PP2B (Hescheler et al., 1988). Microystin-LR is also a potent inhibitor of both PP1 (IC50 1.7 nM) and PP2A (IC50 0.04 nM), while having no effect on PP2C (Honkanen et al., 1990; Takai et al., 1995). 2.2. Protein phosphatase 2B inhibitors Both immunosuppressants, cyclosporin A (Hultsch et al., 1991; Sigal and Dumont, 1992; Walsh et al., 1992) and FK506 (Hultsch et al., 1991; Waschulewski et al., 1993), and the type II pyrethroids cypermethrin, deltamethrin, and fenvalerate (Enz and Pombo Villar, 1997) are all potent inhibitors of PP2B. Cyclosporin A inhibits PP2B activity with an IC50 of approximately 40–200 nM (Hultsch et al., 1991; Sigal and Dumont, 1992; Walsh et al., 1992). Cyclosporin A binds to and inhibits a family of basic cytosolic receptor proteins, cyclophilin A (18 kDa), which are widely expressed in most tissues (Fischer et al., 1989). FK506 (IC50 0.2–3 nM) binds to the rotamase site of FK506 binding protein and inhibits PP2B activity with a greater potency than that for cyclosporin A (Waschulewski et al., 1993). FKBP

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proteins, of which there are five members, have a molecular weight of 12–52 kDa and all possess rotamase activity. Both cyclophilin and FKBPs catalyse cistrans peptidyl-prolyl isomerization and protein folding (Marks, 1996). 2.3. Protein tyrosine phosphatase inhibitors Dephostatin (IC50 7.7 mM) is a competitive inhibitor of PTP, but does not inhibit the activity of the serine/ threonine phosphatase 2A and 2B (Imoto et al., 1993; Fujiwara et al., 1997). Vanadate, at nanomolar concentrations (IC50 2 nM), also inhibits PTP (Swarup et al., 1982; Enz and Pombo Villar, 1997).

3. Role of phosphatases in regulation of non-neuronal cell function 3.1. Phosphatase 1 & 2A Calyculin A has been shown to cause contraction of intact smooth muscle of guinea pig taenia ceci and rat aorta in the presence or absence of extracellular calcium (Ishihara et al., 1989a). Similarly, okadaic acid stimulated contraction of both vascular and intestinal smooth muscle via a Ca21-independent protein kinasedependent mechanism (Shibata et al., 1982; Ozaki et al., 1987; Hirano et al., 1989). In human bronchial smooth muscle, contractile and relaxant responses to okadaic acid have been attributed to effects on myosin phosphatase and Ca21-calmodulin-dependent myosin light chain kinase activity (Savineau and Marthan, 1994). In contrast, okadaic acid failed to induce contraction or relaxation of guinea pig isolated trachea (Harrison et al., 1997). Furthermore, okadaic acid appears to increase the open probability of Ca21-dependent K1 channels in rabbit airway smooth muscle cells and augment the isoprenaline-induced increase in open probability of Ca21dependent K1 channels (Kume et al., 1989), but did not alter the relaxation response to isoprenaline in guinea pig isolated trachea (Harrison et al., 1997). Relatively low concentration of microcystin-LR increases the force of myosin light chain (MLC20) phosphorylation in the femoral artery, while higher concentrations are needed to affect the ileum (Gong et al., 1992). In b-ecin-permeabilized smooth muscle, microcystin-LR, tautomycin, and okadaic acid induced contractions which are similar to Ca21-induced responses (Gong et al., 1992). 3.2. Protein phosphatase 2B Immunosuppression is a consequence of inhibition of calcineurin and the signal transduction pathways which lead to the activation of specific transcription factors (e.g. nuclear factor of activated T cells, NF-AT) involved in IL-2 gene transcription in lymphocytes (Liu, 1993; Ho et al., 1996). The inhibition of PP2B by

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cyclosporin A and FK 506 is thought to be responsible for the inhibition of IgE-dependent degranulation of mast cells and basophils (Hultsch et al., 1991; Stellato et al., 1992; Marone et al., 1993) and exocrine secretion from the pancreas (Waschulewski et al., 1993; Groblewski et al., 1994). Cyclosporin A has been reported to mediate effects that are unrelated to inhibition of calcineurin. Cyclosporin A inhibited the binding of radiolabelled substance P to membrane homogenates of guinea pig lung, indicative of neurokinin-1 antagonism (Gitter et al., 1995). Similarly, the ability of substance P to stabilize IL-2 mRNA in Jurkat cells is inhibited by cyclosporin A (Calvo, 1994). In contrast, cyclosporin A failed to inhibit histamine release from human skin mast cells induced by substance P (Stellato et al., 1992). 3.3. Protein tyrosine phosphatase Vanadate has been demonstrated to induce contraction in tracheal smooth muscle isolated from guinea pig (Nayler and Sparrow, 1983; Cortijo et al., 1993), dog (Lee et al., 1994), monkey and rabbit (Ueda et al., 1985). The mechanism of action for vanadate-induced contraction in guinea pig trachea appears to be via intracellular Ca21 mobilization which is linked to inhibition of Ca21-dependent ATPase activity (Nayler and Sparrow, 1983). In anesthetised guinea pigs, vanadate induces bronchoconstriction (Nayler and Mitchell, 1987). Vanadate has also been shown to evoke formation of inositol 1,4,5-trisphosphate (Bianchini et al., 1993) and enhance protein tyrosine phosphorylation in guinea pig taenia coli smooth muscle (Di Salvo et al., 1993).

protein phosphatase inhibitor attenuated the contractile response of guinea pig isolated bronchus following excitation of nonadrenergic noncholinergic nerves (eNANC), indicating that in this species PP1/2A plays a role in facilitating the release of neuropeptides from airway sensory nerves (Harrison et al., 1997). The inhibitory effect of okadaic acid on the eNANC contractile response was not attributable to a nonspecific affect of this agent, since 1-Nor okadaone, an inactive phosphatase inhibitor, was without action on the eNANC response (Harrison et al., 1997). Interestingly, okadaic acid at concentrations which induce a rise of intracellular Ca21 in rat sensory neurones, failed to alter either the capsaicin-induced rise in Ca21 (Cholewinski et al., 1993) nor the desensitization of these neurones to capsaicin (Docherty et al., 1996), or affect the contraction of guinea pig bronchus to exogenously administered capsaicin (Harrison et al., 1997). The cellular targets of protein phosphatases in airway sensory nerves is not clear at present, although there are several possibilities. Okadaic acid can reduce the activity of N-type calcium channels (Sculptoreanu et al., 1995), which are critical for the release of neuropeptides following depolarization of sensory nerves (Maggi, 1995). Similary, okadaic acid can increase the open probability of calcium activated potassium channels (Kume et al., 1989). The calcium activated potassium channel blocker, charbydotoxin, has been shown to attenuate the ability of various agonists to inhibit the eNANC response (Stretton et al., 1992), implicating this channel in the regulation of neuropeptide release from airway sensory nerves. 4.2. Protein phosphatase 2B

4. Role of phosphatases in regulation of neuronal cell function 4.1. Phosphatase 1 & 2A Okadaic acid is reported to augment acetylcholine release from the neuromuscular junction (Abdul Ghani et al., 1991; Swain et al., 1991). Similarly, calyculin A increased synaptic transmission in the rat hippocampus (Herron and Malenka, 1994). In contrast, okadaic acid decreased evoked but not spontaneous release acetylcholine from rat hippocampus, but tended to increase spontaneous and not evoked release of glutamate (Vickroy et al., 1995). The effect of okadaic acid on acetylcholine release may be related to a reduction in the synthesis of acetylcholine in the tissues (Issa et al., 1996). In our study, okadaic acid was without effect on cholinergic contractile responses in guinea pig trachea (Harrison et al., 1997). Okadaic acid augmented capsaicin, potassium, and bradykinin-induced release of substance P from cultured rat dorsal root ganglion (DRG) neurones (Hingtgen and Vasko, 1994). We have demonstrated that this

Studies have demonstrated that mRNA for cyclophilins and PP2B is localized to nerve cells and widely distributed throughout the brain and spinal cord (Steiner et al., 1992; Dawson et al., 1994; Chen et al., 1995). Moreover, cyclophilin and PP2B appear to be co-localized in neurones (Steiner et al., 1992; Dawson et al., 1994). There are further suggestions that ion channel function is regulated by calcineurin, which is inhibited by both the cyclosporin A-cyclophilin and FK506FKBP complexes (Chad and Eckert, 1986). Furthermore, cyclosporin A-cyclophilin have recently been shown to inhibit capsaicin-evoked desensitization of dorsal root ganglion neurones from rats (Docherty et al., 1996). FK506 and cyclosporin A cause a spontaneous and K1 depolarization-induced release of neurotransmitters from PC12 cells (Steiner et al., 1996). In cultured fetal rat cortices, cyclosporin A (5–20mM) or FK506 (0.1–1 mM) increases the rate of spontaneous neuronal firing, which may be due to enhanced release of glutamate by presynaptic cells and it has been proposed that PP2B acts presynaptically to regulate Ca21-dependent release

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Fig. 1. Summarises the modulatory effect of protein phosphatases on neuropeptide release in sensory nerves. Studies have revealed that cAMP dependent pathways inhibit N-type Ca21 channels and Ca21-activated K1 channels while augmenting L-type Ca21 channels. In guinea-pig airways, okadaic acid inhibits neuropeptide release while FK506 and cyclosporin A (CsA) stimulate release. (1) excitatory action, (2) inhibitory action.

of glutamate (Victor et al., 1995). FK506 (50 mM) inhibited postsynaptic PP2B in adult rat hippocampal slices thereby enhancing synaptic transmission both basally and following stimulation of synapses (Wang and Kelly, 1996). FK506 (10–50 mM) blocked long-term depression expressed in hippocampus in postnatal rats (Mulkey et al., 1994). Similarly, cyclosporin A promoted phosphorylation of sodium channels in rat brain (Chen et al., 1995) and promoted dephosphorylation of dynamin in rat brain terminals (Nichols et al., 1994). GABAA receptor-mediated responses are thought to be regulated by PP2B, and it is assumed that PP2B enhances the ligand-gated ion channels, as both cyclosporin A/ cyclophilin A (14–50 nM) (Boddeke et al., 1996; Martina et al., 1996) and deltamethrin (0.5 mM) attenuate desensitization (Hardwick and Parsons, 1996). We have recently demonstrated that cyclosporin A induced a concentration-dependent contraction of guinea pig isolated bronchus that was antagonized by neurokinin 1 and 2 selective antagonists, abrogated by the vanilloid receptor antagonist, capsazepine, and inhibited following acute desensitization with capsaicin (Harrison et al., 1998). Our results suggest that cyclo-

sporin A may stimulate the vanilloid receptor, although the role of PP2B in this effect was not clear since relatively high concentrations were employed and the cyclosporin H produced a contractile response, albeit to a lesser extent. In contrast, the contractile response to FK506 was unaffected by capsazepine, but inhibited by the n-type Ca21 channel blocker, v-conotoxin (Harrison et al., 1998). The type II class of pyrethroids are extremely potent inhibitors of PP2B (IC50 , 1 nM) and they have been demonstrated to affect voltage-gated Na1 channels. It has been shown that all type II pyrethroids promote the translocation of protein kinase C on an IP3-independent Ca21 triggering site to the plasma membrane, which is thought to increase the mechanisms involved in depolarization or receptor-induced neurotransmitter release (Zurgil et al., 1986; Nichols et al., 1987; Enan and Matsumura, 1992). Deltamethrin, at extremely low concentration, causes stimulation of neurotransmitter release in isolated rat brain synaptosomes (Clark and Brooks, 1989). Similarly, deltamethrin and fenvalerate cause depolarization of nerve membranes (Lund and Narahasi, 1983; Salgado et al., 1983). This is consistent with the

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idea that they have the ability to slow down the closing time of sodium channels as seen in crayfish neurones (Salgado et al., 1989). Deltamethrin has been shown to cause a rise in protein phosphorylation within synaptic process which was accompanied by a sharp rise in intracellular Ca21 concentrations (Enan et al., 1996). This rise in Ca21 was also observed when external Ca21 was replaced by Ba21, suggesting the release of Ca21 from intracellular stores. Cypermethrin (1 nM) suppressed GABAA responses in guinea pig hippocampal neurones (Stelzer and Shi, 1994). 4.3. Protein tyrosine phosphatase In rat synaptosomal preparation, deltamethrin (0.01 nM) stimulated neurotransmitter release (Clark and Brooks, 1989). Similarly, vanadate augmented contractile response to capsaicin in guinea pig isolated trachea but not to exogenously administered substance P, histamine, or acetylcholine, suggesting a prejunctional action on neuropeptide release (Nayler, 1988). 5. Conclusion It would appear from the evidence provided that protein phosphatases play an important role in regulating neuronal processes (Figure 1). Clearly further study are required to elucidate the mechanism by which phosphatases regulate neuronal responses in the airways, and the discovery of more potent and selective inhibitors will greatly facilitate this process. Furthermore, such tools will allow investigators to probe the effect of altering neuronal activity in the context of airway inflammation. References Abdul Ghani, M., Kravitz, E.A., Meiri, H., Rahamimoff, R., 1991. Protein phosphatase inhibitor okadaic acid enhances transmitter release at neuromuscular junctions. Proc Natl Acad Sci USA 88, 1803–1807. Abe, H., Tamura, S., Kondo, H., 1992. Localization of mRNA for protein phosphatase 2C in the brain of adult rats. Brain Res Mol Brain Res 13, 283–288. Adcock, J.J., Garland, L.G., 1993. The contribution of sensory reflexes and ‘hyperalgesia’ to airway hyperresponsiveness. In: Page, C.P., Gardiner, P.J. (Eds.), Airway hyper-responsiveness: is it really important for asthma? (1st ed.). Blackwell, Oxford, pp. 234– 255. An, J., Carmichael, W.W., 1994. Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins. Toxicon 32, 1495– 1507. Arino, J., Woon, C.W., Brautigan, D.L., Miller, T.B. Jr., Johnson, G.L., 1988. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes. Proc Natl Acad Sci USA 85, 4252–4256. Aroca, P., Bottaro, D.P., Ishibashi, T., Aaronson, S.A., Santos, E., 1995. Human dual specificity phosphatase VHR activates maturation promotion factor and triggers meiotic maturation in Xenopus oocytes. J Biol Chem 270, 14229–14234.

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