Assessment of signal transduction mechanisms regulating airway smooth muscle contractility.

Assessment of signal transduction airway smooth muscle contractility CRAIG

M. SCHRAMM

AND

MICHAEL

mechanisms

regulating

M. GRUNSTEIN

Division of Pulmonary Medicine, Joseph Stokes, Jr., Research Institute, Children’s Hospital of Philadelphia, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104 Schramm, Craig M., and Michael M. Grunstein. Assessmentof signaltransduction mechanismsregulating airway smooth musclecontractility. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L119-Ll39, 1992.Agonist-receptor interactions regulate airway smooth muscletone through activation of guanine nucleotide binding proteins (G proteins) which are coupledto second-messenger pathways that mediatechangesin the tissue’s contractile state. Various methodshave been applied to identify the structure/function characteristics of G proteins and their role in signal transduction in airway smooth muscle,including the use of exotoxins, nonhydrolyzable analogsof guanosinetriphosphate(GTP), antibodiesto purified G proteins, and membranereconstitution studies. In elucidating mechanismsof airway smooth musclerelaxation, considerableprogresshas been made in identifying the molecular basis for receptor/G protein coupling and other regulatory processesleading to both the activation and downregulation of the adenylate cyclase/adenosine3’ ,5’ -cyclic monophosphate system.Further, with respectto airway smoothmusclecontraction, various approacheshave been usedto evaluate the role of membranephosphoinositide turnover and the mechanismsof action of the bifurcating signal transduction pathways associatedwith the production and metabolismof inositol1,4&trisphosphate and l,%diacylglycerol, and activation of protein kinase C. This review identifies much of the information gainedto date on the abovesignaltransduction pathways, with an emphasisplacedon various methodological approachesused to determine membraneand transmembrane signalingprocessesin airway smooth muscle. transmembrane signaling; cyclic nucleotides; protein phosphorylation; phospholipaseC; methodology; airway contractility IN RECENT YEARS, the identification

of molecular components that participate in transmembrane signaling pathways has provided fundamental new insights into the mechanisms that regulate the cellular response to receptor activation with hormones, drugs, and neurotransmitters. Airway smooth muscle contains a host of pharmacologically distinct receptors which are coupled to contraction or relaxation of the tissue in response to agonist stimulation. In the case of bronchoconstrictor agonists, receptor activation typically elicits membrane phosphatidylinositol turnover that results in the formation of the second messengers, l,%diacylglycerol, which activates protein kinase C, and inositol 1,4,&trisphosphate, which mobilizes intracellular Ca2+. Both the mobilization of Ca2+ and activation of protein kinase C play crucial roles in initiating and acutely modulating the intensity and duration of the airway smooth muscle contractile response. In the case of bronchodilator agonists, on the other hand, receptor activation is typically coupled to an enhanced accumulation of the second messenger, adenosine 3’,5’-cyclic monophosphate (CAMP) which, through activation of CAMP-dependent 1040-0605/92

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protein kinase, induces the phosphorylation of specific proteins leading to airway smooth muscle relaxation. For activation of both of these functionally distinct signal transduction pathways, the agonist-receptor complexes interact with specific guanine nucleotide binding proteins (G proteins) which, in turn, modulate the enzymes regulating the production of their respective second messengers. This review addresses much of the information gained to date on the above principal transmembrane signaling mechanisms coupled to airway smooth muscle relaxation and contraction, with an emphasis placed on the methodological approaches and techniques used to examine the role of G proteins and mechanisms of upand downregulation of second-messenger accumulation and function. Limitations of space preclude a full treatment of other important processes regulating the airway smooth muscle contractile response including ion channel conductance, intracellular Ca2+ regulation, and contractile protein activation, all of which have been recently reviewed (18, 32, 44, 126, 167, 190, 197, 209, 214). The review begins with a general overview of the structural and functional characteristics of G proteins,

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followed by an account of approaches used to identify the role of G proteins in regulating airway smooth muscle tone. Subsequently, the discussion focuses on the mechanisms of activation and desensitization of the adenylate cyclase/cAMP transduction pathway and the methods used to identify the role of this signaling pathway in regulating airway smooth muscle relaxation. Finally, the role and mechanisms of action of the phosphoinositollinked signal transduction system are discussed, including approaches used to evaluate the production and metabolism of inositol 1,4,5trisphosphate, the role of the protein kinase C signaling pathway, and mechanisms of desensitization of this phospholipase C-coupled system. IDENTIFICATION IN TRANSMEMBRANE

OF

G

PROTEINS SIGNALING

As noted above, G proteins play a pivotal role in transmembrane signaling pathways. Although in some signaling systems the same membrane protein recognizes the extracellular stimulus and effects the intracellular response (e.g., tyrosine kinase receptors), the majority of receptors are linked with their effector systems via specific G proteins. In their intermediary roles, G proteins are responsible for the translational convergence of multiple stimuli into the cell’s relatively fewer effector systems. In addition, G proteins are capable of modulating and amplifying receptor-mediated generation of such second messengers as the cyclic nucleotides, CAMP and cGMP, inositol 1,4,5trisphosphate, and Ca2+. This intermediary regulation determines, at least in part, the cell’s integrated response to agonist stimulation. Although the role of G proteins in transmembrane signaling has been extensively reviewed (15, 19, 89, 128, 150, 202), the following is an account of various approaches used to examine certain structural and functional characteristics of G proteins as they relate to smooth muscle contractility. All of the well-characterized G proteins appear to be heterotrimers consisting of an a-subunit that binds guanine nucleotides and possesses intrinsic GTPase activity, and tightly associated p- and y-subunits that anchor the a-subunit to the cytoplasmic surface of the cell membrane (150). Specificity of receptor-effector action appears to be largely determined by structural and functional differences in the a-subunits. To date, eight different mammalian G proteins have been biochemically characterized and associated with cu-subunit-specific signal transduction pathways (Table 1). Five or six additional a-subunits have been identified by complementary DNAs (138, 194), but their cellular functions remain to be elucidated. Of interest, one of these novel a-subunits, tentatively called ag, is relatively concentrated in rat lung (161). Although distinct forms of the ,& and ysubunits have also been identified, they appear to be functionally equivalent (19). No information is presently available as to whether the latter subtypes regulate cellular distribution of the G proteins or interaction of the &complex with specific cu-subunits, although as, can discriminate between transducin &- and By-complexes derived from nonretinal G proteins (33).

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In its resting state, each G protein exists with GDP bound to the nucleotide binding site of the a-subunit. Association of an agonist to its receptor results in the enhanced release of a-bound GDP and creation of an agonist-receptor conformation with a high affinity for the cvPy-heterotrimer in which the nucleotide site is “open.” This ternary complex of agonist-receptor-G protein is transitory; entry of GTP into the nucleotide binding site of the a-subunit triggers the dissociation of the @y-complex from the receptor and separation of aGTP from the fly-subunits. The “active” wGTP subunit is then able to interact with a particular effector enzyme or ion channel to modulate second-messenger synthesis or ion exchange, respectively. Hydrolysis of the bound GTP by intrinsic GTPase activity in the a-subunit terminates its interaction with the effector, and the a-GDP reassociates with the fly-complex to form a heterotrimer ready for activation by another receptor molecule. lModuLation of GTPase. Agonists interact with receptors associated with a GDP-liganded (i.e., inactivated) G protein with higher affinity than with receptors not in contact with such a G protein. In contrast, pure antagonists do not demonstrate such differential affinity. Thus ligand binding studies in a variety of systems demonstrate that dose-response curves of agonist displacement of [3H]antagonist binding are nonideal, with Hill coefficients significantly less than 1. Addition of poorly hydrolyzed analogues of GTP [e.g., guanosine 5’-[&y-imino]triphosphate (Gpp(NH)p) or guanosine 5’-O-(3-thiotriphosphate) [ (GTP?S)] to the binding conditions produces a stable cw-GTP-analogue complex, thereby reducing the ability of agonists, but not of antagonists, to compete for [ 3H] antagonist binding. Accordingly, in the presence of stable GTP analogues, the shapes of agonist displacement curves are close to ideal. The latter observations of guanine nucleotide sensitivity of agonist binding provided the basis for the first identification of G protein-associated receptors. In recent applications of the above technique, both muscarinic cholinergic and leukotriene receptors have been linked to G proteins in the lung. In bovine tracheal smooth muscle, the muscarinic agonist oxotremorine was shown to displace [ 3H] quinuclidinyl benzilate (QNB) binding in a manner consistent with agonist binding to high- and low-affinity sites having Kn values of 3.8 nM and 2.2 PM, respectively (136). Addition of GTPyS shifted the high-affinity sites to low affinity, suggesting that the high-affinity sites represented muscarinic receptors coupled to G protein(s) (136). Similarly, in competition experiments against the selective LTD, antagonist, [3H]-ICI 198,615, in guinea pig lung membranes, 1 ,uM Gpp(NH)p caused a significant rightward shift of the inhibition by agonists LTD4, LTE4, and YM-17690 but not by different antagonists (36). Stable analogues of GTP may also be administered to permeabilized cells in order to examine the contribution of G proteins to the cellular response (e.g., second-messenger generation), independent of specific receptor activation. Gpp(NH)p and GTPyS are capable of stimulating phosphoinositide metabolism independent of any agonist in many cell types (166) and have been shown to potentiate agonist-mediated phosphoinositide hydrolysis


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Table

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1. Receptor, effector, second messenger, and toxin specificities of biochemically defined G protein a-subunits a-Subunit

ai1 ai %3

Receptors

,&Adrenergic Hz-histamine Pg& VIP Others M2 and M4 cholinergic Adenosine ar-Adrenergic

at1

M1 and M3 cholinergic HI-histamine Prostaglandins Tachykinins Endothelin Others Odorants Photons

at2

Photons

Qolf

Effecters

Signaling

Toxin

Adenylate cyclase L-type Ca2+ channels

T CAMP T Ca2+ Influx

Cholera

Adenylase cyclase Phospholipase C Phospholipase A2

Pertussis

K+ channels Phospholipase C K+ channels Ca2+ channels

J CAMP T I&, DAG Arachidonate release T K+ current T I&, DAG j’ K+ current 4 Ca2+ current

Adenylate cyclase cGMP phosphodiesterase

t CAMP (olfaction) 4 cGMP (vision)

cGMP phosphodiesterase

4 cGMP (color vision) (? T I&, DAG)

Cholera Cholera Pertussis Cholera Pertussis None

Pertussis

cholinergic) (? phospholipase C) others) Characteristics of the currently identified G protein a-subunits that couple the above receptors to cellular effecters and second messengers. The corresponding bacterial toxin sensitivities are noted. PgE2, prostaglandin E2; VIP, vasoactive intestinal peptide; M, muscarinic molecular species. See text for other abbreviations.

in membrane preparations, including from human lung concentration and GTPase activity of G, compared with fibroblasts (170). GTP (0.1 mM) has been shown to other membrane G proteins. Thus agonist-receptor-stimaugment contractions with phenylephrine and carbachol ulated hydrolysis of GTP by activated G, may be lost in in guinea pig portal vein and ileum, respectively, and this the background basal rates of h.ydrolysis occurring in the potentiation was ablated by prior addition of the sta- other more prevalent and more active G proteins present ble GDP analogue, guanosine 5’-O-(2-thiodiphosphate) (142). (GDPPS: 1 mM) (113). Whereas the effect of GTPyS is Use of bacterial toxins. Exotoxins isolated from Vibrio essentially irreversible, G proteins may be reversibly cholerae and Bordetella pertussis have proved to be adactivated by fluoroaluminate complexes, such as AlF,, ditional invaluable tools in the characterization of G which circumvent the need for GDP dissociation by proteins, particularly those associated with the adenylate binding alongside GDP and mimicking the terminal cyclase signaling system. Pertussis toxin (formerly called phosphate group of GTP (14). Fluoroaluminate can stimislet activating protein) contains an ADP-ribosyltransulate phosphotidylinositol metabolism and can inhibit ferase subunit that catalyzes the transfer of the ADPhormonal stimulation of CAMP in many cells and memribose moiety of NAD+ to the a-subunit of certain G branes. Curiously, however, although it is capable of proteins. Early experiments with this toxin demonstimulating adenylate cyclase in membranes, fluoroalustrated an enhancement of GTP activation of adenylate minate does not appear to be capable of stimulating G, cyclase in rat C6 glioma cells, concomitant with the ADPin intact cells (95). ribosylation of a 41-kDa membrane-associated protein As noted above, agonist-receptor stimulated exchange (107). Such initial studies suggested that pertussis toxin inactivated Gi, thereby diminishing tonic or agonistof GTP for GDP in the a-subunit’s guanine nucleotide binding site is followed by hydrolysis of the nucleotide specific inhibition of adenylate cyclase activity. It has by the intrinsic GTPase activity of the a-subunit. Ac- subsequently been demonstrated that, by ribosylating a cordingly, measurement of enhanced GTPase activity in cysteine residue on the a-subunit, pertussis toxin blocks membrane preparations in response to agonist binding the ability of receptors to interact with and activate provides another simple and direct approach to assess several G proteins, including Gi (inhibitor of adenylate the interaction of a receptor with its G protein. This cyclase) and at least one form of G, (activator of phosapproach, like the above radioligand and functional pholipases C and A,). Pertussis toxin has been shown to attenuate muscamethods, however, is limited in its ability to specifically identify or quantify the G proteins mediating specific rinic cholinergic modulation of CAMP in canine tracheal effects. Additionally, it is often impossible to demonsmooth muscle. In this regard, acetylcholine was found CAMP strate receptor stimulation of GTPase activity, even to decrease basal and isoproterenol-stimulated though other evidence may exist to indicate that the accumulation in a dose-dependent manner. In contrast to control tissues, wherein 100 PM acetylcholine was receptor interacts with a G-protein signaling system found to decrease isoproterenol-stimulated CAMP con(142). The latter limitation is particularly evident with receptor-G, interactions, because of the relatively lower tent by about 34%, acetylcholine reduced CAMP by only Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 9, 2019.

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8% in tracheal smooth muscle treated with 10 pg/ml pertussis toxin (176). Thus cholinergic functional antagonism of ,&adrenergic function in canine tracheal smooth muscle appears to involve a pertussis toxin-sensitive, CAMP-inhibitory G protein (i.e., Gi). In contrast, pertussis toxin was not found to exert any significant effect on high-affinity oxotremorine binding to bovine trachealis membranes (136). Taken together, these observations suggest that there exists interspecies variation in the coupling of G proteins to airway smooth muscle muscarinic cholinergic receptors and/or that cholinergic receptors may interact with two distinct classes of G proteins (i.e., pertussis-sensitive and pertussis-insensitive) in this tissue. Like pertussis toxin, cholera toxin also contains an ADP-ribosyltransferase. In the presence of a second protein, referred to as ADP-ribosylation factor (ARF) (lOl), cholera toxin catalyzes the transfer of ADP-ribose from NAD+ to an arginine residue of a, close to the guanine nucleotide binding domain. Such ribosylation inhibits the intrinsic GTPase activity of the subunit (34), rendering G, in a sustained activated state and eliminating the requirement of receptor participation in G protein activation. Thus treatment with cholera toxin can maximally activate adenylate cyclase in a tissue or membrane preparation. Recently, however, effects of cholera toxin have been observed that were not mimicked by CAMP (94). The latter observation suggests either that the toxin may affect another presently uncharacterized G protein or that G, may exert effects independent from adenylate cyclase stimulation. The polypeptide substrates for either cholera or pertussis toxin may be identified and relatively quantified by gel electrophoresis using [““PINAD+ as the ADP source. In the presence of guanine nucleotides, cholera toxin promotes the specific incorporation of radiolabel into a,, with an apparent molecular mass of either 45 or 52 kDa. The latter polypeptides are probably not distinct as-subunits; rather, the two forms appear to represent differentially spliced products derived from a single gene (142). In contrast, pertussis toxin is able to catalyze 32P labeling of several polypeptides, with apparent molecular masses between 39 and 41 kDa. Incubation of canine tracheal smooth muscle with pertussis toxin (12.5 pg/ ml) for 21 h was shown to label a protein of apparent molecular mass of 40 kDa (176). Resolution of the different pertussis toxin-sensitive G proteins in one-dimensional sodium dodecyl sulfate (SDS)/polyacrylamide gels is often poor, but may be improved with two-dimensional electrophoresis (142). One particular complication, however, may confound the use of bacterial toxin-catalyzed ADP-ribosylation as a means of quantitatively comparing G proteins in different tissues or at different maturational stages. In rat brain, levels of both Gil and G, were found to markedly decrease with age when measured by pertussis toxinmediated ADP-ribosylation, but were later found to increase ontogenetically when assessed by Western blotting with specific antibodies. This discrepancy was subsequently resolved with the demonstration that brain NAD-glycohydrolase significantly increases with age and, hence, substantially degrades the [““PINAD+ in

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older tissue (143). Thus if less than maximal ADPribosylation occurs in the tissue (i.e., PLC-7 (171). Whether any of these PL-C isozymes that have been purified are responsible for receptor-mediated, G protein-dependent stimulation of PIP2 hydrolysis in situ remains to be established. Assessment of phosphoinositide metabolism. The early studies of phosphoinositide metabolism relied on chromatographic means to separate the individual phospholipids and inositol phosphates, followed by determination of radioisotope incorporation into each fraction. Membrane phospholipids are extracted from cell pellets with chloroform/methanol/concentrated HCl and are separated by one- or two-dimensional thin-layer chromatography (TLC) on silica gel high-performance thin-layer plates impregnated with oxalate (137). One-dimensional

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chromatography adequately resolves PIPB, PIP, and PI; however, two-dimensional chromatography is necessary to also separate the phosphatidic acid (1). Separated lipids are visualized by iodine vapor, which can detect about l-10 pg of lipid, and identified by comparison with authentic standards (137). Simultaneously, the aqueous tissue extract can be used for determination of inositol phosphates by various techniques, of which the most widely applied is anion-exchange chromatography. Sequentially washing a formate-column (e.g., Dowex 1 x 8 or BioRad AG 1 x 8) with 0.1 N formic acid and 0.2 M, 0.5-0.6 M, and 1.0 M ammonium formate in 0.1 M formic acid will elute inositol, IP, IP2, and IP3, respectively (1, 137) When tissues are labeled with 32P, isotopic equilibrium of IP, PIP, and PIP2 with the ATP pool is reached within 30 min; with [myo-3H] inositol labeling, 90 min is required (1). The presence of carbachol during the labeling period stimulates incorporation of [3H]inositol into a receptor-sensitive phosphoinositide pool, thereby allowing steady-state conditions to prevail during subsequent agonist incubations and ablating large agonist-induced changes in specific radioactivity in the phosphoinositides (40). Thereafter, stimulation with Ca2+-mobilizing agonists results in a rapid accumulation of radiolabeled IP3, and loss of PIP2 within 5 to 15 s, but no significant increase in IP until about 2 min, suggesting that the primary plasma lipid utilized by these receptors is PIP2 (1). Such studies often employ lithium to selectively inhibit inositol phosphate phosphatase, the enzyme that hydrolyses IP back to inositol (77). Using these methods, Baron et al. (3) first demonstrated that cholinergic contraction of canine trachealis was associated with a decline in the membrane phosphatidylinositol (PI) pool, an increase in the phosphatidic acid and diacylglyercol pools, and an increased incorporation of 32P into PI. Subsequent investigators demonstrated that concentrations of PIP and PIP2 also fell rapidly in bovine trachealis stimulated with carbachol (43,200) and that in tracheal smooth muscle, as in other tissues, PIP2 appeared to be the major membrane phosphoinositide hydrolyzed by hormone-activated PL-C (200). The addition of 5 mM LiCl to the assay conditions has been shown to result in a 5- to lo-fold enhancement of carbachol-mediated IP production, twofold enhancement of IP2 production, and negligible changes in IP3 (41, 70). Further assays of tracheal smooth muscle labeled with [3H]inositol and stimulated with agonists in the presence of 5-10 mM LiCl have shown increased [3H]IP accumulation in response to other muscarinic agonists (70, l4O), electrical field stimulation (141), histamine (76), serotonin (124), bradykinin (40), and leukotrienes D, and E4 (145). Moreover, a direct relationship was found in bovine trachealis between IP accumulation and contraction for a variety of muscarinic agonists (140). In cruder preparations from guinea pig airways, similar results have been obtained with the tachykinins, substance P and neurokinin A and B (71). Despite its usefulness in assays of phosphoinositide metabolism, several concerns have recently arisen regarding the inclusion of Li+ in such studies. First, Li+ has been shown to primarily increase levels of the inac-


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tive isomer 1,3,4-IP3 rather than the active product 1,4,5- of stimulation, cellular 1,4,5-IP3 levels are back to their IP3, thereby enhancing total IP3 content but not the basal values. A similar, but lesser, response was observed physiological response (27). Second, in the absence of with histamine (41). agonist, Li+ alone leads to large accumulations of all the In comparison to the above 1,4,5-IP3 responses to inositol phosphates over time (1). Third, Li+ may also contractile agonists, we have recently demonstrated that affect sites other than IP phosphatase, in light of the endothelin (ET-l) elicits a similar increase in 1,4,5-IP3 observation that in the presence of carbachol, Li+ pro- levels in mature rabbit tracheal smooth muscle, from duces concentrationand time-dependent inhibition of basal values of 14.0 t 1.2 (SE) pmol/mg protein to 30.8 IP3 accumulation that it is not related to receptor desen- t 4.2 (SE) with 0.1 PM endothelin (72). In contrast to sitization (4). the transient nature of the IP3 response to carbachol and Determination of 1,4,5-IP3 accumulation. Overwhelmhistamine, however, we have observed that the endotheing evidence has emerged from a multitude of cell types lin-elicited increase in cellular ‘1,4,5-IP3 is sustained for confirming that 1,4,5-IP3 is the molecule that links recep- up to 10 min (72). The mechanism(s) underlying the tor-activated phosphoinositide breakdown to Ca2+ mo- persistent endothelin-specific IP3 generation are curbilization from intracellular stores (reviewed in 10). The rently under investigation, and may involve 1) the unapplication of 1,4,5-IP3 to permeabilized canine tracheal usually slow and possibly incomplete dissociation of ET smooth muscle results in 45Ca2’ release and the develfrom its receptor (86); 2) potential interaction with a opment of tension (80). The release of Ca2+ in permenovel G protein displaying low GTPase activity [e.g., G, abilized cells is rapid and occurs at less than micromolar (69)], which would permit prolonged G protein activation concentrations of 1,4,5-IP3 (166). Additionally, injection following interaction with the ET receptor; 3) endotheof 1,4,5-IP3 into intact cells has been shown to mimic lin-specific inhibition of the 3’-kinase or 5’-phosphatase receptor-mediated events in hepatocytes (31) and Xeno- enzymes linked to ‘1,4,5-IP3 degradation; and/or 4) enpus oocytes (159). Moreover, the time course for 1,4,5dothelin-specific enhancement of membrane PIP2 conIP3 formation paralleled secretagogue-induced Ca2+ re- tent, a factor which can also limit IP3 production (9). lease in pancreatic acinar cells (195). Last, blockade of The latter potential mechanism is supported by the PL-C-mediated 1,4,5-IP3 production with neomycin was independent observations that the supply of PIP2 is shown to inhibit Ca2+ release by agonists (195). enhanced by PK-C stimulation (51) and that endothelin Although measuring total IP accumulation provides is a potent stimulator of PK-C activation in airway evidence of agonist-mediated phosphoinositide hydrolysmooth muscle (72). sis, it is an indirect assay of 1,4,5-IP3 formation. If, in High-affinity binding sites for radiolabeled 1,4,5-IP3 some tissues or under certain conditions, PL-C hydrohave been identified in a number of tissues. With purilyses varying amounts of PI and PIP into IP and IP2, fication and cDNA isolation from rat cerebellar Purkinje respectively, the total IP accumulation may significantly cells, the 1,4,5-IP3 receptor has been characterized as a misrepresent the IP3 response. Even if IP3 is separated membrane glycoprotein consisting of 2,749 amino acids chromatographically from the other inositol phosphates, with a deduced molecular mass of 313 kDa and high the kinetics of the total IP3 response may not reflect affinity for 1,4,5-IP3 (& 21.7 nM) (64). Some evidence 1,4,5-IP3 production because of the agonist-stimulated suggests that the protein may exist as a tetramer in vivo into lipid vesicles has demonstrated formation of an inactive IP3 isomer, 1,3,4-IP3. Either of (53). Reconstitution two methods may be employed to separate the two IP3 that the purified protein possesses both the 1,4,5-IP3 compounds: 1) high-performance liquid chromatography recognition sites and Ca2+ channel activity (56). The using a Partisil SAX analytical column fitted with a receptor is a major substrate for CAMP-dependent by which inhibits both agonist precolumn containing Whatman pellicular anion ex- PK-A, phosphorylation change resin and eluted with an ammonium phosphate binding and 1,4,5-IP3-mediated Ca2+ release (53). The gradient (41, 50); or 2) enzymatic degradation of the characteristics of 1,4,5-IP3 binding to its receptor in membrane fractions from bovine trachealis have been inactive 1,3,4-IP3 by crude rat brain supernatant under Mg2+-free conditions (109). With the use of either demonstrated to be of high affinity (& 3.8 t 0.2 nM), method, it can be shown that, within 60 s of carbachol saturable (B,,, 1,003 t 170 fmol/mg protein), specific for d-1,4,5-IP3 vs. l-1,4,5-IP3 or &1-1,3,4-IP3, and inhibstimulation, 1,3,4-IP3 accounts for ~80% of the [3H]IP3 accumulation in prelabeled bovine tracheal smooth musitable by heparin with an ICsO of 7.6 + 1.0 pg/ml (42). cle (41). Assessment of 1,4,5-IP3 metabolism. Because PIP2 conBecause of the above difficulties in quantitating tinues to be hydrolyzed for 30-60 min in the presence of changes in 1,4,5-IP3 content, several groups of investicarbachol (40, 200), the brisk decline in 1,4,5-IP3 levels must be attributed to rapid degradation of 1,4,5-IP3. A gators have developed a radioreceptor assay method based on the specific binding of the 1,4,5-trisphosphate series of specific kinases and phosphatases terminates isomer to its putative intracellular receptor (21,41, 160). the signaling activities of 1,4,5-IP3 and salvages inositol By exploiting the presence of a high-affinity binding site, for lipid resynthesis (reviewed in Shears 183). Principal Chilvers and co-workers have been able to detect as little among these enzymes are the 1,4,5-IP3-3’-kinase and 5’as 20 fmol of 1,4,5-IP3 with the 1,4,5-[3H]IP3 radiorecepphosphatase, the products of which are 1,3,4,5-IP4 and tor assay (41). With this technique, carbachol has been 1,4-IP2, respectively. 1,3,4,5-IP4 may also function as a shown to elicit a rapid but transient increase in 1,4,5-IP3 second messenger to regulate Ca2+ influx and/or Ca2+ stores and, in assocontent in bovine trachealis, from a resting level of 12.9 translocation between intracellular -+ 0.8 to 27.0 t 1.5 pmol/mg protein at 5 s. Within 30 s ciation with 1,4,5-IP3, may sustain the Ca2+ signal during Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 9, 2019.

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prolonged stimulation (reviewed in 53). In the brain, 5’phosphatase exists as at least two isozymes: type I 5’phosphatase has a molecular mass of 66 kDa and degrades both 1,4,5-IP3 and 1,3,4,5-IP4 and type II 5’phosphatase has a molecular mass of 160 kDa and is more specific for 1,4,5-IP3 (53). Activities of the 3’-kinase and 5’-phosphatase enzymes may be determined from the breakdown of [3H]1,4,5-IP3 by crude enzyme preparations from tissue homogenates in reaction mixtures containing l-4 mM MgClz for the 5’-phosphatase or lo-20 mM MgCl,, lo20 mM ATP, and 5 mM 2,3-diphosphoglycerate (an inhibitor of IP3 5’-phosphatase) for the 3’-kinase (82, 146). After termination of the reactions, the inositol phosphates may be separated either by high-voltage electrophoresis in sodium oxalate buffer (82) or by ammonium formate elution from anion-exchange columns (146). Identity of the products 1,3,4,5-IP4 and 1,4-IP2, may be verified by comigration with authentic standards on a Partisil SAX HPLC column with a 0.01-1.0 M ammonium phosphate gradient (50). Our recent data indicate that the K, and Vmax values for the 3’-kinase in rabbit tracheal smooth muscle homogenates, amounting to 5.0 PM and 0.14 nmol min. mg protein-‘, respectively (172a), are similar to those reported for 3’-kinase activity in the rat brain [i.e., values of 3.6 PM and 2.5 nmol . min. mg protein-l, respectively (82)]. The K, values for 3’-kinase and 5’-phosphatase recently determined in crude enzyme preparations from rabbit tracheal segments amounted to 5.0 and 95 PM, respectively (172a), and were comparable to the values reported in other tissues (183). A priori, these differences in K, favor the phosphorylation route for 1,4,5-IP3 metabolism. Whether 1,4,5-IP3 degradation occurs via this route in airway smooth muscle will also depend, however, on the relative Vmax values for phosphorylation and dephosphorylation, the intracellular concentration of 1,4,5IP3 and, possibly, the spatial distribution of the 3’-kinase and 5’-phosphatase within the cell (183). Moreover, acute modulation of the activities of the latter enzymes can provide additional regulatory mechanisms for the fine control of cellular responses to PL-C activation. In this regard, acute negative feedback inhibition of intracellular 1,4,5-IP3 levels may be provided by Ca2+-calmodulin activation of 3’-kinase (13) and possibly 5’-phosphatase (178) and protein kinase C activation of the 3’kinase (12,93) and perhaps 5’-phosphatase (reviewed in ref. 183).

concentrations. The importance of PK-C activation in signal transduction is well documented, and a host of physiological functions have been ascribed to the enzyme, including modulation of secretion, exocytosis, ion conductance, smooth muscle contraction, receptor regulation, gene expression, and cell proliferation (1, 16, 17, 110,152-154,166,168). The myriad effects of PK-C activation are likely attributed to the existence of at least seven different isozymes comprising two general classes of PK-C, with molecular masses of 68-84 kDa. The a-, PI-, PII-, and ysubspecies are characterized as having four conserved (C&J and five variable (V,-V,) regions, whereas the second group of 6-, E-, and PMA > PDD to this approach, however, is that although usually >90% (48) > diC:8 (125). Guinea pig parenchymal contractions of the original specific activity of PK-C can be downDownloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 9, 2019.

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regulated with chronic exposure, the residual PK-C may be constitutively activated by the phorbol and still be capable of modulating cellular responses. Additionally, it has been recently demonstrated that the E-PK-C isoform is not down-regulated by chronic phorbol ester exposure (196). Use of PK-C Inhibitors. An alternative approach to investigate the role of PK-C in a given response has been the use of “selective” inhibitors of the kinase. Three general classes of inhibitors are available, including those that 1) interact with the enzyme’s activating site; 2) interact with the catalytic domain; and 3) interact with the regulatory ‘site. Of the activating site inhibitors, those that block diacylglycerol/phorbol ester binding (e.g., 1-0-alkyl-2-0-methylglycerol [AMG], calphostin C) represent more specific antagonists for PK-C. Inhibitors of Ca2+/phospholipid binding (e.g., dibucaine, trifluoroperazine, polymyxin B, adriamycin) and inhibitors of all three binding sites (e.g., sphingosine, aminoacridines) are also capable of competing with calmodulin and, accordingly, are equally effective inhibitors of CaM-dependent protein kinases (91). Although the catalytic domain inhibitors are somewhat more potent PK-C antagonists, because of the homology in kinase ATP binding sites, these compounds also show varying selectivity for PK-C relative to other kinases (see Table 3). In contrast, highly specific “pseudosubstrate” peptides have been synthesized that irreversibly bind to the region responsible for maintaining PK-C inactivity in the absence of activator molecules. Such peptides are very potent inhibitors of PK-C in vitro; however, because of their size and polarity, they are impermeable to intact cells and, hence, are ineffective as pharmacologic agents. Using the above approaches of PK-C “depletion” and “selective inhibition,” investigators have further elucidated the role of the kinase in modulating contractile responses in airway smooth muscle. Pretreatment with PMA (1 PM x 45 min) or with the PK-C inhibitor, H-7 (10 PM), has been shown to inhibit 2-pyridylethylamineinduced contractions in guinea pig lung parenchymal strips by -30 and 20%, respectively (125). Similarly, administration of 100 PM H-7 was found to inhibit the rabbit tracheal contractile response to acetylcholine by Table

3. Protein

kinase inhibitors Ki, PM

Compounds

Ref. cGMP-PK

HA 1004 K-252a K-252a H-7 Staurosporin Staurosporin UCN-01 Calphostin

CAMP-PK

1.3

2.3 0.20 0.018 3.0 0.12 0.008 0.042

0.020 5.8

>50

kynEe

150

97

PK-C

40 0.47 0.025 6.0 0.01 0.003 0.004 0.05

CaM-PK

0.30

0.04

(85) (49) (104) (85) WV (174) (174) (201)

List of reported inhibitors of protein kinase C, including their affinity constants (K;) for different kinases. HA 1004, N-(2guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride; K-252a, (8R*, 9S*, 1lS*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,lO-tetrahydro-8,ll-epoxy-lH,8H,l1H-2,7b,lla-triazadibenzo(a,g)cycloocta(cde)trinden-l-one; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride. See text for other abbreviations.

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-36% (72). In contrast, PK-C inhibitors were found to dramatically inhibit tracheal contractions elicited with endothelin by 92.5% with H-7, 60.3% with staurosporin and 31.9% with HA 1004. Moreover, the order of potency of the latter antagonists in reversing endothelin-induced contractions was staurosporin >> H-7 > HA 1004, in parallel to their rank order in potency for inhibiting PK-C in cell-free extracts (Table 3) (72). Trandocation of PK-C. Although careful use of phorbol esters and kinase antagonists can provide indirect evidence for the role of PK-C in mediating certain cellular responses, assay methods exist for more direct measurement of PK-C activation following receptor stimulation. On activation, PK-C is translocated from its resting cytosolic position to a site associated with the plasma membrane. Thus evidence for involvement of PK-C in mediating the responses to extracellular agents is provided by a decrease in cytosolic and increase in membrane-associated PK-C activity. Detergents such as Nonidet P-40 or Triton X-100 may be used to solubilize the PK-C bound to pelleted cell membrane fractions, followed by DEAE-cellulose chromatographic fractionation of both cytosolic and membrane samples to remove endogenous components and excess detergent that interfere with the PK-C assay (206). PK-C activity may then be determined by measuring the transfer of 32P from [y32P]ATP to a suitable substrate (usually histone H, or 111s) in buffer containing Ca2+, Mg2+, and a diacylglycerol, and subtracting the amount of nonspecific 32P incorporation in the absence of phosphatidylserine from incorporation in the presence of phosphatidylserine (206). Alternately, native-membrane associated PK-C activity may be measured without solubilization by assaying 32P phosphorylation of a PK-C-specific cytosolic 85-kDa protein substrate, separated, and quantitated on a 10% polyacrylamide gel (37). Such techniques have not as yet been applied in studies on airway smooth muscle. DESENSITIZATION C-COUPLED SIGNAL

OF

PHOSPHOLIPASE TRANSDUCTION

Considerably less is known about desensitization of phospholipase C-coupled responses than has been elucidated with regard to the adenylate-cyclase system. All existing evidence, however, points to comparable mechanisms of homologous and heterologous desensitization in this pathway. Parallel mechanisms of homologous desensitization have been demonstrated in a few nonairway smooth muscle types. Indeed, it has been shown that within lo20 min of exposure to norepinephrine, -40% of aladrenergic receptors become inaccessible for [3H]prazosin binding at 4°C on intact DDT,MF-2 hamster smooth muscle cells, whereas binding to fragmented cells is unchanged. The latter findings are consistent with the notion that a-adrenergic agonists are able to induce sequestration of al-adrenoreceptors (61). Desensitization of DDTIMF-2 cells with the al-agonist phenylephrine is not associated with translocation of PARK activity, however, suggesting that ,&ARK does not interact with cyladrenergic receptors and that PARK translocation is likely not activated by protein kinase C (193). In con-


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trast, short-term (i.e., 10 min) exposure of chick heart tissue to acetylcholine has been shown to result in a threefold decrease in the tissue’s subsequent sensitivity to carbachol (117). This desensitization has been associated with time- and dose-dependent phosphorylation of the muscarinic receptors and corresponding decreases in high-affinity agonist binding. Phosphorylation of the cardiac muscarinic receptor was unaffected by the activation of protein kinase C or CAMP- or cGMP-dependent protein kinase, or by inhibition of Ca2+/calmodulin-dependent protein kinase (117). This muscarinic receptor phosphorylation was later determined to be mediated by the same receptor kinase that mediates P-adrenoreceptor phosphorylation, i.e., @ARK (116). The potential role of PARK in regulating other nonadrenergic contractile receptors (e.g., muscarinic cholinergic, H1 histaminergic) remains to be established. At least four potential mechanisms have been proposed for heterologous desensitization of the Ca2+-mobilizing response by PK-C based on findings that 1) PK-C activation down-regulates Ca2+ -mobilizing receptors, including al-adrenergic (122) and angiotensin (22) receptors in vascular smooth muscle and HI-histamine receptors in hamster DDTIMF-2 smooth muscle cells (144); 2) PK-C activation inhibits phospholipase C activation with HI-histamine receptor stimulation in cultured canine airway smooth muscle cells (149); 3) PK-C activates the 3’-kinase (12,94) and 5’-phosphatase enzymes (183) mediating 1,4,5-IP3 breakdown [indeed, it has been speculated that the prompt reversal of 1,4,5-IP3 accumulation in the presence of continued PIP2 metabolism allows for the ongoing formation of 1,Sdiacylglycerol and PK-C activation without persistent 1,4,5-IP3-driven intracellular Ca2+ release (43)]; and 4) PK-C may phosphorylate both myosin light chain (151) and its kinase (92), thereby inhibiting the Ca2+ -induced actin-myosin interaction involved in smooth muscle contraction. With exception to the demonstration that PK-C inhibits H1-histaminergic stimulation of PL-C activity (149), little is known about the mechanisms of down-regulation of Ca2+-mobilizing receptors in airway smooth muscle. The future application of many of the above techniques and the adaptation of methods developed in evaluating the regulation of the adenylate cyclase system should provide new information concerning the regulation of the Ca2+-mobilizing signaling pathway in airway smooth muscle. CONCLUSION

In recent years, significant progress has been made toward understanding the membrane signal transduction pathways involved in regulating airway smooth muscle tone. The molecular and biochemical processes underlying receptor-coupled control of second-messenger production and function are complex and, as emphasized in the above discussion, a number of fundamental issues remain unresolved. In this regard, some of the important areas requiring further investigation include identification of the molecular structure/function relationships of various receptors coupled to airway smooth muscle contractility; determination of the molecular identities and roles of different G proteins; assessment of the mecha-

REVIEW

nisms of action of 1,4,5-IP3, protein kinase C, and cyclicnucleotide-dependent protein kinases in regulating Ca2+ signaling, myosin light chain phosphorylation, and contractile protein function, and determination of the roles and mechanisms of action of other products of membrane phosphoinositide hydrolysis in modulating the initiation, intensity, and duration of the contractile response. With the continued application of the methodological approaches described herein, and the incorporation of new techniques (e.g., site-directed mutagenesis, use of sitedirected antibodies, application of transgenic cellular and whole animal models, etc.), one can look forward to significant new insights which will be gained from the above areas of investigation into the receptor-effector interactions regulating airway smooth muscle tone. We thank P. Lorenski for secretarial assistance. This work was supported by the National Heart, Lung, and Blood Institute Grants HL-43285 to C. M. Schramm and HL-31467 and HL45063 to M. M. Grunstein. Address for reprint requests: M. M. Grunstein, Division of Pulmonary Medicine, Childrens Hospital of Philadelphia, Univ. of Pennsylvania, School of Medicine, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. REFERENCES 1. Abdel-Latif, A. A. Calcium-mobilizing receptors, phosphoinositides, and the generation of second messengers. P&urm&oZ. Reu. 38: 227-272,1986. 2.

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