Biochimica et Biophysica Acta, 1176 (1993) 313-320 313 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

BBAMCR 13367

Adenylyl cyclase in lung from hypersensitive guinea pig displays increased responsiveness to guanine nucleotides

and isoprenaline: the role of the G proteins G s and Gi

Michael Grady, Patricia A. Stevens, Susan Pyne and Nigel Pyne Department of Physiology and Pharmacology, University of Strathclyde, Glasgow (UK)

(Received 6 May 1992) (Revised manuscript received 17 November 1992)

Key words: Adenylyl cyclase; Hypersensitivity; G protein; Protein kinase C; (Lung); (Guinea pig)

Basal adenylyl cyclase activity in lung membranes isolated from hypersensitive guinea pigs was increased and more sensitive to stimulation by isoprenaline, GTP and GppNHp when compared to adenylyl cyclase in lung membranes isolated from normal healthy guinea pigs. Maximal forskolin-stimulated adenylyl cyclase activity was unaltered. There was no change in the immunological quantitative amounts of either a subunits of the G proteins Gil I and G~ (Go, Gii and Gilll were not present). Maximal pertussis-toxin- and cholera-toxin-catalyzed ADP-ribosylation of Gia and Gs~ respectively were not significantly altered. The addition of purified protein kinase C to isolated lung membranes resulted in the phosphorylation of the a subunit of G~ (stoichiometry was 0.53 mol of 32p incorporated/mol of Gs~). Addition of protein kinase C to lung membranes isolated from hypersensitive guinea pigs was equally effective at catalysing the phosphorylation of the a subunit of G s. GppNHp-stimulated and basal adenylyl cyclase activity was also enhanced in isolated tracheal smooth-muscle membranes from hypersensitive guinea pigs. These results suggest that hypersensitive reactions are associated with the improved coupling of the stimulatory G protein (G~) with adenylyl cyclase.

Introduction

Hypersensitivity (type-l-immediate reaction) is a condition brought about by the excessive production of immunoglobulins (IgG, IgE), the subsequent release of inflammatory mediators [1,2] from mast cells, macro- phages and eosinophils and the development of ana- phylaxis. Examples of inflammatory mediators released are PGF2~, LTD4, LTC 4 and histamine. These have the ability to promote contraction of airway smooth- muscle and elicit bronchoconstriction. These inflamma- tory mediators are also released in the early phase of asthma and, therefore the induction of hypersensitivity in guinea pigs has served as an animal model for asthma.

The hyperreactivity associated with these reactions may induce alterations in the responsiveness of the lung cells and alveolar macrophages to various inflam-

Correspondence to: N. Pyne, Department of Physiology and Phar- macology, University of Strathclyde, 204 George St., Glasgow, UK. Abbreviations: GppNHp, guanosine 5'-(/3-imido)triphosphate; GTP, guanosine 5'-triphosphate.

matory mediators. Recent studies [3,4] assessing the changes in the responsiveness of both lung cells and alveolar macrophages to agonist stimulation have yielded conflicting reports. For instance, Gadd and Bhoola [3], demonstrated that lung homogenates con- tain elevated basal and GTP-stimulated adenylyl cy- clase activities. However, the magnitude of GppNHp- stimulated adenylyl cyclase activity was unaltered. These workers have therefore suggested that the in- trinsic GTPase activity of the a subunit of G s may be impaired by hypersensitivity reactions. In contrast, /3- adrenoceptor-stimulated adenylyl cyclase activity was reduced; this apparently due to an inability of the stimulatory guanine nucleotide binding regulatory pro- tein (Gs) to functionally couple with the receptor. In contrast to these studies, alveolar macrophage mem- brane adenylyl cyclase isolated from hypersensitive guinea pigs exhibited enhanced responsiveness to stim- ulants of G~, such as cholera toxin and prostanoids, e.g., PGE l [4]. Furthermore, antigen challenge elicited an enhanced GppNHp-stimulated adenylyl cyclase ac- tivity in subsequently prepared membranes [4].

In this paper we describe an enhanced /3-adrenoc- eptor stimulation of lung adenylyl cyclase activity asso-

314

ciated with hypersensitivity. This may occur as a conse- quence of a modulation of G s and adenylyl cyclase coupling, since both GTP and GppNHp also appear to elicit larger activations of adenylyl cyclase activity. However, the increased coupling was not due to im- paired GTPase activity of the a subunit of G s, elevated expression of Gs, , or reduced expression of G~,. Pro- tein kinase C is implicated in the sensitization mecha- nism [5], and also in the modulation of G i and G s function [6,7]. However, whilst protein kinase C cat- alyzes the phosphorylation of Gs~ in lung membranes, this reaction is not modulated by hypersensitivity. We also present evidence that a similar enhanced basal and GppNHp-stimulated adenylyl cyclase activities oc- curs in tracheal smooth-muscle membranes from hypersensitive animals.

Materials and Methods

Biochemicals were from Boehringer-Mannheim (Germany), whilst chemicals and a-actin antibody were from Sigma (UK). Isotopes such as [a-32p]ATP and [3H]cAMP were from Amersham International (UK). [32 P]NAD + was from Dupont (USA). Anti-G s antibod- ies (MG1) were raised by us to the C-terminal de- capeptide of Gs, (RMHLRGYELL). Anti-G i antibod- ies (NP1) were raised by us to the C-terminal decapep- tide of G m (KNNLKDCGLF). Anti-G i antibodies (SG1), anti-G o antisera (IM10), anti-Gil (IC1) and anti- Gin I (I3B) anti-sera were kind gifts from Dr. G. Milli- gan (University of Glasgow). Recombinant 45-kDa Gs, , (purified from cDNA transfected E. coli lysates ac- cording to Graziano et al, [8]), was a kind gift from Dr. M. Freissmuth (University of Vienna). Horseradish- peroxidase-linked anti-rabbit antibodies were from the Scottish Antibody Production Unit. Purified protein kinase C was purchased from Lipidex (USA).

Preparation of membranes from lung and trachea. Routinely one lung was removed from a male Dunkin- Hartley guinea pig (300 g) by careful dissection. The lung was perfused with gassed Krebs-Hensleit (pH 7.4), and then homogenized in three volumes of buffer A, containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1 mM PMSF and 2 mM benzamidine, at 4°C using 4-5 up-and-down strokes of a Turrex homog- enizer. The resulting homogenate was filtered through cheesecloth and then subjected to centrifugation at 48 000 X g for 20 rain at 4°C in a Beckman SW 55.2 rotor. The supernatant was discarded and the pellet resuspended in buffer A and recentrifuged as before. The resulting pellet was again resuspended in buffer A and stored at -20°C. Membranes were prepared from the isolated tracheal smooth-muscle strips in an identi- cal fashion. Protein determination were performed ac- cording to Bradford [9].

Cell culture. Preparations of the primary cultures of airway smooth-muscle was achieved by removing the trachea from guinea pigs and dissecting free the carti- lage and the epitheluim to leave the tracheal smooth- muscle strip. This was performed under sterile condi- tions in an air-flow cabinet. The smooth muscle was titrated in 4 ml of Dulbecco's Modified Eagle's medium (DME) containing collagenase (Type II, 1 mg/ml), elastase (type IV, 0.2 mg/ml) and soya bean trypsin inhibitor (50/xg/ml) before transfer into a further 1 ml of the medium and incubation at 37°C for 1.5-2 h until fully digested. The cell suspension was then diluted (1 : 5) with DME containing 10% (v/v) foetal calf serum (FCS) and 10% (v/v) donor horse serum (DHS) and divided between two 25-cm 2 collagen-coated tissue cul- ture flasks which were incubated at 37°C in air /CO 2 (95:5%, v/v). The medium was replaced with DME containing 10% (v/v) FCS and 10% (v/v) DHS after 48 h. The cells were routinely passaged twice, using trypsin, prior to experimentation. Cells were grown to confluence and routinely used 13-15 days after the initial preparation, and were confirmed as smooth muscle by the presence of a-actin using a smooth- muscle-specific monoclonal antibody raised to a-actin [10].

Incubation of cells. Ceils were placed in DME con- taining 1% FCS and 1% DHS, 24 h before use. The cells were then washed twice in gassed (95 : 5% air:CO 2) Krebs Hensleit (pH 7.4), and finally incubated in 10 ml of Krebs Hensleit. The cells were then challenged with PMA (10 -7 M) for 15 min at 37°C. In some cases the cells were given an 18h pre-incubation with pertussis toxin (0.1 /zg/ml). After incubation, the cells were washed twice in Krebs Hensleit buffer (pH 7.4), prior to harvesting in this buffer. The cells were pelleted using bench centrifugation at 1000 rpm for 2 min, and membrane fractions prepared by homogenizing the pel- lets in 2 ml of ice-cold buffer A. Homogenates were centrifuged at 48000 ×g for 20 min at 4°C and the subsequent pellet resuspended in buffer B, containing 10 mM Hepes (pH 7), and 10 mM fl-glycerophosphate and taken for adenylyl cyclase assay.

Induction of hypersensitivity. Routinely, 100 p.g of ovalbumin and 100 ng of AI(OH) 3 was injected into healthy guinea pigs according to Anderson [11]. The animals were left for 14 days prior to being killed. Hypersensitivity was assessed by testing the ovalbumin induced contraction of ileum smooth muscle. This was achieved by mounting the smooth muscle (connected to an isometric transducer recorder) under 1-g tension in 10 ml of Krebs-Hensleit (pH 7.4), which was constantly gassed. Sham-injected animals (Al(OH)3-injected) were compared and showed no characteristic contraction in response to ovalbumin.

'Pre-activation' of G s by GppNHp. The lung and tracheal smooth-muscle membranes (100/xg) were cen-

trifuged at 48000 x g at 4°C for 20 min and resus- pended in buffer C containing 10 mM Tris-HC1 (pH 7.4), 5 mM MgC12 and 1 mM DTI'. To 100 /zl of membranes was added an equal volume of GppNHp (final concentration, 100 /xM), and the samples were incubated at 37°C for 1 h. After this time, the samples were centrifuged for 10 min at 15 000 × g at 4°C. The pellet was resuspended in buffer B and taken for the adenylyl cyclase assay. Under these conditions, acti- vated G s will accumulate due to quasi-irreversible bind- ing of GppNHp.

Measurement of adenylyl cyclase activity. Adenylyl cyclase activity was measured according to Saloman et al. [12]. This involved combining membranes from ei- ther lung or tracheal smooth muscle (20-30 mg) with an activation cocktail containing (final concentration) 12.5 mM Tris-HC1 (pH 7.4), 2.5 mM'MgSO4, 0.4 mM cyclic AMP, 2.5 mM creatine phosphate, 1.5 units of creatine kinase, 100/xM ATP, 7.5 mM KCI and 30 mM sucrose. Routinely 1000000 cpm of [a-32p]ATP was added to the assay. In some instances final concentra- tions of GppNHp (10 -4 M), forskolin (10 -4 M), iso- prenaline (10 -6 M) and/or GTP (10 -4 M) were added to the reaction cocktail. The incubation was performed at 37°C for 20 min and terminated by the addition of 40 mM ATP and boiling for 2 min. Samples were then centrifuged at 15000 × g for 10 min and the super- natants removed. To each sample supernatant was added [3H]cyclic AMP (5000 cpm). This procedure enabled the measurement of recovered [a-32p]cyclic AMP from Dowex WH ÷ and alumina column chro- matography. Recovered [a-32p]cyclic AMP was quanti- fied by liquid scintillation counting. Experiments were performed where less than 10% of the substrate was used and under conditions of linear rate of formation of product.

Toxin-catalyzed ADP-ribosylation of G s and G i. Cholera-toxin-catalyzed ADP-ribosylation of lung membranes was performed according to Heyworth et al. [13]. This involved combining membranes (30 /~g) with an ADP-ribosylation cocktail containing (final concentration) 150 mM potassuim phosphate (pH 7.6), 3 mM DT-F, 5 /xM CaCI2, 1 mM MgC12, 7.5 mM thymidine and to this 7.5/~Ci [32p]NAD, (20/xM) was added. To initiate the incubation, cholera toxin (66 /~g/ml) was added. This had been pre-activated by combining it with an equal volume of 50 mM DTI" and incubating the sample for 1 h at room temperature. Ribosylation incubations were performed at 37°C for 2 h after which they were terminated by the addition of TCA (final concentration, 6%, w/v) and deoxycholate (final concentration 0.01%, w/v). The samples were centrifuged at 15 000 × g for 10 min at 4°C in a Beck- man microfuge. The resulting pellet was resuspended in 10/xl of 1 M Tris-HCl, and 20 ~1 of Laemmli buffer and these were boiled for 5 min prior to SDS-PAGE

315

(10% acrylamide). Pertussis-toxin-catalyzed ADP-ribo- sylation was performed according to Pyne et al. [14]. Membranes (30 /xg) were combined with buffer con- taining (final concentrations) 30 mM thymidine, 1 mM ATP, 80 mM potassium phosphate (pH 7.5), 20 mM arginine, 1 mM MgCI 2, and to this was added 7.5/xCi of 20/xM [32p]NAD +. These were then combined with pre-activated pertussis toxin (1.33 tzg/assay) and incu- bated at 37°C for 2 h. Pre-activation of pertussis toxin was performed in an identical manner to cholera toxin. In all cases the 2-h incubation allowed maximal ADP- ribosylation.

After electrophoresis, the gels were fixed in 10% TCA for 30 min and dried prior to autoradiography at -20°C for 72 h. Location of the ADP-ribosylated polypeptides was followed by quantification by Cerenkov counting of the radioactive gel chips.

Western blotting. Lung membranes (200-300 mg) were subjected to SDS-PAGE, and transferred to nitrocellulose. After transfer, the sheets were blocked in 3% (w/v) gelatin and buffer D containing 10 mM Tris-HC1 (pH 7.4), and 0.9% NaC1 at 37°C for 2 h. After washing with distilled water, the sheets were incubated with an anti-G s or anti-G i antibody for 12 h at 30°C. Routinely a 1:200 dilution of anti-sera was used. After this incubation, the sheets were washed in buffer D containing Tween-20 (0.05%, v/v) and then in buffer D alone. The sheets were then incubated with a horseradish-linked anti-rabbit antibody (1:200 dilu- tion), for 2 h at room temperature and then washed as before. The sheets were then reacted with 1 ml of O-dianisidine (10 mg/ml), 10/xl of hydrogen peroxide (30%, v/v) and 40 ml of 10 mM Tris-HCl (pH 7.4), in order to locate the immunoreactive bands. Quantifica- tion was achieved using reflective densitometry to mea- sure the optical density of immunoreactive peptides at 420nm. Optical density was linearly related to the amount of protein blotted (0-300 ~xg).

Phosphorylation incubation in isolated lung mem- branes. Lung membranes (100 /xg protein) were cen- trifuged at 15 000 × g for 10 min at 4°C in a Hettich microcentrifuge and the pellets resuspended in buffer B. 100 txl of membranes were then combined with an activation cocktail containing (final concentrations) 12.5 mM Hepes (pH 7), 1.25 mM MgCI 2, 0.375 mM CaCI2, 12.5 /zM ATP, [y-3zp]ATP (5 /~Ci/assay), 0.5 mM PMSF and 5 mM benzamidine. To initiate the incuba- tion, 0.25 pmol/min of purified brain protein-kinase-C activity, containing a mixture of isoenzymes (specific activity = 2.5 nmol/min per mg of protein kinase C, see Ref. 6) was added. The total incubation volume was 200 ~1 and the samples were incubated at 37°C for 20 min. Incubations were terminated by adding stau- rosporine (final concentration, 1 ~M). After this, the samples were combined with 135 /~l of 10 mM phos- phate-buffered saline (pH 7.4), 125/xl of buffer B plus

316

4% lubrol (v/v) and with anti-G s antibodies (final dilution 1: 25) to a final incubation volume of 500/~1. These samples were incubated at 4°C for 12 h. After this time, the samples were centrifuged at 4°C in a Hettich microcentrifuge at 15000 × g for 10 min and to the resulting supernatant, 20 ~1 of immunoprecip- ±tin (10%, w/v suspension) was added. Samples were incubated for 2 h at 4°C after which they were cen- trifuged at 4°C at 15000 × g for 2 min. The immun- precipitin pellets were each washed three times in 1 ml of phosphate-buffered saline (pH 7.4), and centrifuged as before. The final immunoprecipitin pellet was resus- pended in 20 /zl of Laemmli buffer, boiled and sub- jected to SDS-PAGE. The polyacrylamide gels were fixed in 10% (w/v) TCA for 1 h, dried and autoradio- graphed at - 20°C for 24 h. Quantification of 32p-phos- phorylated Gs~ was by Cerenkov counting of excised 32p-polypeptide bands.

Results

Adenylyl cyclase activity in guinea-pig lung and tracheal membranes

The 'pre-activation' procedure was employed in or- der to allow accumulation of activated G s under condi- tions where adenylyl cyclase activity is limited by the lack of ATP. Basal adenylyl cyclase activities in lung and tracheal smooth-muscle membranes from hyper- sensitive animals were approx. 2-fold and 1.4-fold larger in magnitude, when compared to adenylyl cyclase activ- ities in normal guinea pigs (Table I). The fold activa- tion of adenylyl cyclase activity with GppNHp does vary due to the low basal adenylyl cyclase that is measured as recovered [32p]cyclic AMP. However, un- der identical assay conditions we observe reproducible differences between controls and matched hypersensi- tive GppNHp-stimulated adenylyl cyclase specific act±v-

TABLE I

Effect of GppNHp upon adenylyl cyclase activity in lung and tracheal smooth-muscle membranes

Adenylyl cyclase activity was measured according to Materials and Methods. The concentration of GppNHp was 10 -4 M. Results of adenylyl cylase activity are expressed as pmol/min per mg of protein. These results are means±S.D. (n = 3 triplicate assays) for a repre- sentative experiment performed upon four different membrane preparations.

Treatment Adenylyl cylase activity (pmol/min per mg)

normal hypersensitive

untreated lung 7.89± 0.8 15.78± 2.6 10 -4 M GppNHp 339 ± 39 702 ± 100 untreated tracheal

smooth muscle 8 ± 0.2 10.9 ± 0.97 10-4M GppNHp 79.6 ± 1.3 167 _+ 3.5

5 0 -

4 0 '

o ~ 3 0 ' ~ .

• ~ 2 0

~ o

I0

0 I i ! i

O - 9 - 8 - 7 - 6 - 5 - 4 - 3

Fig. 1. Effect of GppNHp upon adenylyl cyclase activity in lung membranes from normal and hypersensitive animals. A graph illus- trating the dose-relationship of GppNHp-stimulated adenylyl cyclase activity in lung membranes from normal ( , ) and hypersensitive ([]) guinea pigs. This is a representative result of an experiment per- formed three times. Concentrations of GppNHp used ranged from 10-10_10 4 M. Results, expressed as means_+S.D for triplicate assays, are represented as -fold stimulations of basal adenylyl cyclase

activity in lung membranes isolated from healthy guinea pigs.

±ties (Table I, Fig. 1). Concentration-dependent activa- tion of adenylyl cyclase activity in isolated membranes from control animals, with GppNHp, displayed an EC50 of approx. 0.5 /~M GppNHp (Fig. 1). Hypersensitivity apparently induces an enhanced maximal activation of adenylyl cyclase activity, with no change in the ECs0 for GppNHp (Fig. 1). The 'pre-activation' procedure leads to loss of adenylyl cyclase activity in lung mem- branes. However, this loss of activity was approximately the same in both sets of membrane preparations (70% for normal membranes, and 62% for hypersensitive membranes, Tables I and II).

GTP, GppNHp and isoprenaline (added directly to the assay) resulted in a similar enhanced stimulation of adenylyl cyclase activity in lung membranes from hypersensitive animals (Table II and Fig. 2). Basal adenylyl cyclase activity in lung membranes from hypersensitive animals was 1.5-fold larger than in con- trol lung (Table II). However, forskolin-stimulated adenylyl cyclase activities, at maximal activating con- centrations, were virtually identical (Fig. 2). Maximal activation elicited by forskolin was markedly lower than that observed at maximal activating concentra- tions of GppNHp (Fig. 2). GppNHp (10 -8 M) en- hanced sub-maximal forskolin-stimulated adenylyl cy- clase activity (-fold activation of adenylyl cyclase activ- ity: 1.45 forskolin, 10 -6 M, 3.73 GppNHp, 10 -8 M, 9.1 GppNHp plus forskolin). We were unable to show any action of a number of inhibitory agonists of adenylyl

TABLE II

Isoprenaline-stimulated adenylyl cyclase activity in lung membranes from normal and hypersensitive animals

Adenylyl cyclase activity was measured according to the methods. Results are expressed as p m o i / m i n / m g of protein. Results are mean + S.D. for a single representative results (n = 3, triplicate as- says) of an experimental performed three times with different mem- brane preparations.

Adenylyl cylase (pmol /min per mg)

normal hypersensitive

Control 27 + 0.82 41 + 1 GTP(10 -4 M) 115 + 5 196.2+ 5.8 Isoprenaline (10-6 M) 46.8 _+ 7.6 94.8_+ 19.6 GTP + isoprenaline 161.2_+ 28 316 + 34

cyclase activity as assessed by addition of inhibitory agonist to membranes, and measurement of either GTP- or forskolin-stimulated adenylyl cyclase activity (data not shown).

Immunological assessment of the amount of G s and G i in membranes

In order to establish if the enhanced GppNHp- stimulated adenylyl cyclase activity was due to an in- creased expression of Gs,,, we subjected membranes to Western blotting with an anti-Gs~ antibody (MG1). We consistently observed similar immunoreactivity levels of the Gs, ~ peptide (control 100%, hypersensitive 107 + 9%, n = 3, Fig. 4). This same anti-serum immunode- tected the a subunit of recombinant G~ (Fig. 3), indi-

Q

"6 r - > , o u

20

~ 10

" 0

lTll Ifir 1 2 3 4 5 6

I ]/'7:¸

'-v"

7 8

Fig. 2. Effect of forskolin and GppNHp upon adenylyl cyclase activity in membranes from hypersensitive and normal animals. The basal (1,2), forskolin 10 -5 M (3,4), 10 -4 M (5,6) and GppNHp 10 4 M (7,8) stimulated adenylyl cyclase activity in membranes prepared from normal (1,3,5,7) and hypersensitive (2,4,6,8) animals are repre- sented as -fold increases from basal adenylyl cyclase activity in lung membranes isolated from normal guinea pigs (1). Results are ex- pressed as means+ S.D (n = 3, triplicate assays) and is a representa- tive experiment performed three times upon different membrane

preparations.

317

Fig. 3. Identification of the a subunit of G s in membranes from lung using peptide-directed antibodies. A Western blot showing the pres- ence of a subunits of G s in membranes from lung. The detection of G s was achieved using an anti-Gs antiserum. Lane 1, 0.1 ~g of recombinant Gs,,; lane 2, lung membranes (300 ~g protein) of normal guinea pig; lane 3 lung membranes (300/~g) of hypersensitive guinea pig. Molecular-weight markers are shown, and the position of Gs, is marked with an arrow. This is a typical result from an

experiment performed three times.

cating that the polypeptide identified in lung mem- branes was the a subunit of G~. We were also unable to show any significant alteration in the amount of G i (control 100%, hypersensitive 120 + 13%, n --- 4) using either SG1 or NP1 antiserum (Fig. 4). No Go, Gii or Giii! could be detected using IM10, IC1 or I3B anti- sera, even though these polypeptides could be immuno- logically detected in brain membranes (data not shown).

Cholera and pertussis-toxin-catalyzed ADP-ribosylation of G s in lung membranes from normal and hypersensitive guinea pigs

Cholera-toxin-catalyzed the ADP-ribosylation of the 45-kDa a subunit of Gs in lung membranes, since no [3zp]ADP-ribosylated polypeptides were detected in the absence of toxin (data not shown). There was no significant difference in maximal cholera-toxin-cata- lyzed ADP-ribosylation of Gs, in lung membranes from normal and hypersensitive guinea pigs (amounts of Gs based upon maximal cholera toxin-catalyzed ADP-ribo- sylation were 0.35 pmol/mg protein from normal ani- mals, and 0.31 pmol/mg protein in membranes from

318

Fig. 4. Immunological analysis of G i in lung membranes from hyper- sensitive and normal guinea pig. A Western blot of lung membranes (300/~g), showing the presence of Gi, ~ subunit. Lane 1, membranes from lung of normal guinea pig; lane 2, membranes from lung of hypersensitive guinea pig. Molecular-weight markers are shown, and the position of Gia is marked with an arrow. This is a typical result

from an experiment performed three times.

hypersensitive animals). Maximal pertussis-toxin-cata- lyzed ADP-ribosylation of Gi~ H was not altered in either set of membranes (amount of G~ based upon maximal pertussis-toxin-catalyzed ADP-ribosylation was 0.1 pmol/mg protein).

Phosphorylation of lung G s by protein kinase C Addition of purified brain protein kinase C to lung

membranes catalyzed the phosphorylation of the a subunit of G s. Less than 10% of phosphorylated Gs, was isolated when protein kinase C was not added to the membrane fraction, suggesting endogenous kinase activity directed toward G~ is evident (data not shown). No phosphorylated polypeptides were immunoprecip- itated in the molecular mass range of 39-42 kDa corresponding to the a subunits of Gi, Go, G z and Gq (Fig. 5). Furthermore, no phosphorylated Gs, was im- munoprecipitated using a pre-immune serum (Fig. 5). The antiserum also successfully immunoprecipitated phosphorylated recombinant Gs, (data not shown), with approx. 40% of the Gsd being immunoprecipitated at a 1:25 dilution of antibody. These experiments confirm that the a subunit of G~ is a substrate for protein kinase C in isolated membranes. The stoichiometry of phosphorylation was determined to be 0.53 mol of 32p incorporated/mol of Gs~ (n = 3). This was calculated using a value of 0.35 pmol of G J m g protein and 300 cpm 32p incorporated into Gs , /mg of lung membrane protein.

In order to establish whether the enhanced coupling between G s and adenylyl cyclase in lung membranes

Fig. 5. Phosphorylation of Gs, , by protein kinase C in lung membranes from normal and hypersensitive guinea pig. A typical autoradiogram showing the protein-kinase-C-catalyzed phosphorylation of the a subunit of G s in lung membranes. In lane 1, normal membranes with pre-immune serum; lane 2, normal lung membranes; lane 3, hypersensitive lung membranes. Molecular-weight markers are shown, and the

position of G s is indicated. This is a typical result from an experiment performed three times.

319

TABLE III

Changes in adenylyl cyclase activity in PMA-challenged tracheal smooth-muscle cells: effect of pertussis toxin

Adenylyl cyclase activity measurements were performed according to Materials and Methods. Cells were challenged with the indicated agonist and subsequent membranes prepared. This is a representative result of an experiment performed at least four times with different cell preparations. Results are expressed as means + S.D. (n = 3, triplicate assay). Fold increases are expressed relative to either basal adenylyl cyclase activity in untreated cells, or GppNHp-stimulated adenylyl cyclase activity in untreated cells.

Treatment Adenylyl cyclase activity (pmol /min per mg) Fold

- GppNHp + GppNHp (10 -4 M) (basal) (GppNHp)

untreated control 26.75 + 3.15 143.3 + 5.6 1 1 PMA (10 -7 M) 49.8 + 1.84 322.7_+6.7 1.85 2.25

pertussis toxin control 51.7 ± 2.7 251.3 + 2.1 1.9 1.75 PMA (10 -7 M) 62.9_+ 1.1 * 391.0-+4.7 * 2.35 2.75

* P < 0.005 for comparison with corresponding untreated PMA-challenged - GppNHP and + GppNHp adenylyl cyclase activities.

from hypersensitive animals was due to an in-vivo protein-kinase-C-catalyzed phosphorylation of G s, pu- rified protein kinase C was added to lung membranes from both normal and hypersensitive animals and the extent of phosphorylation of Gs~ quantified. No appar- ent change in phosphorylation state was detected (Fig. 5).

Effect of PMA challenge of tracheal smooth-muscle cells upon adenylyl cyclase response in isolated membranes

Challenge of tracheal smooth-muscle cells with PMA (10 -7 M) elicited an increase in basal and GppNHp- stimulated adenylyl cyclase activity (1.85- and 2.25-fold increase respectively in PMA challenged cells com- pared to unchallenged cells, Table III). PMA challenge routinely lead to a larger increase in GppNHp-stimu- lated adenylyl cyclase activity when compared to basal activity. Pertussis-toxin-treatment of smooth-muscle cells, and inactivation of G i via irreversible ADP-ribo- sylation, also resulted in enhanced GppNHp-stimu- lated adenylyl cyclase activity in isolated membrane fractions (1.75-fold increase in GppNHp-stimulated adenylyl cyclase activity, Table III). This latter result suggests that G~ can tonically inhibit adenylyl cyclase activity in this membrane preparation. Furthermore, the challenge of pertussis toxin-treated cells with PMA resulted in a 2.35- and 2.75-fold stimulation of basal and GppNHp-stimulated adenylyl cyclase activities re- spectively (Table liD.

Discussion

We have demonstrated that basal and GppNHp- stimulated adenylyl cyclase activities of lung mem- branes from hypersensitive guinea pigs were enhanced and adenylyl cyclase was more sensitive to activation by both isoprenaline and GTP. The observation that iso- prenaline alone was capable of stimulating adenylyl cyclase activity indicates that sufficient endogenous

GTP is available to drive the activation of adenylyl cyclase, and may therefore suggest that basal adenylyl cyclase activity is composed of both endogenous GTP- activated and non-activated components. The lesion in adenylyl cyclase function, associated with hypersensi- tivity, is not confined to the lung, with a similar en- hanced GppNHp-stimulated adenylyl cyclase activity in tracheal smooth-muscle. Our studies confirm conclu- sions derived from similar studies that have used anti- gen-challenged alveolar macrophages [4].

Since no apparent change in the amount of Gs~ was detectable in lung, it is unlikely that increased expres- sion of this G protein can account for the enhanced adenylyl cyclase activities. The possibility that GTPase activity is ameliorated is unlikely, since both GTP- stimulated and GppNHp-stimulated adenylyl cyclase activities are identically affected by hypersensitivity. An increase in the number of adenylyl cyclase molecules that can be activated by GTP-bound Gs~ is also unlikely, since forskolin-stimulated adenylyl cy- clase activity was unaltered. However, maximal forskolin-stimulated adenylyl cyclase activity was lower in magnitude than corresponding maximal GppNHp- activated adenylyl cyclase activity. Activation of adeny- lyl cyclase by forskolin is apparently dependent upon an association of adenylyl cyclase and Gs, since the reconstitution of Gs into $49 cyc- lymphoma cells markedly enhanced forskolin-stimulated adenylyl cy- clase activity [15], and we have also demonstrated that sub-maximal forskolin-dependent activation can be en- hanced by GppNHp. Therefore, the lung may contain a substantial pool of G~ that is directly associated with adenylyl cyclase and which has a kinetically slow disso- ciation rate. Thus, only the coupling between freely available G~ and adenylyl cyclase may be modulated by hypersensitivity reactions.

The elevation of basal and GppNHp-stimulated adenylyl cyclase activity may be a consequence of a protein-kinase-C-catalyzed phosphorylation and inacti-

320

vation of G i and thus a consequential removal of tonic inhibition. In this regard, there is substantial evidence to support the notion that protein kinase C catalyzes the phosphorylation and inactivation of Gi, this having been observed in hepatocytes, $49 lymphoma cells and pre-monocytic U937 cells [14-18]. Furthermore, PMA challenge of tracheal smooth-muscle cells elicits an increase in membrane bound basal and GppNHp- stimulated adenylyl cyclase activity, which is partially ameliorated by pre-treatment with pertussis toxin. Al- ternatively, as in platelets and erythrocytes [19,20], the protein kinase C-catalyzed phosphorylation of adenylyl cyclase may be the predominant mechanism of regula- tion, with this phosphorylation event either activating adenylyl cyclase activity or abrogating its interaction with G i. The extent of phosphorylation of Gs, was not significantly altered in membranes from hypersensitive animals and this provides indirect evidence that the G s is unlikely to be already phosphorylated by protein kinase C in vivo.

The maximal pertussis-toxin- and cholera-toxin- catalyzed ADP-ribosylation of Gi, ~ and Gs,~ were unal- tered by hypersensitivity. /33, subunits of G proteins can markedly increase the ability of the a subunit of G s and G i to act as substrates for cholera-toxin- and pertussis-toxin-catalyzed ADP-ribosylation respec- tively, and can also inhibit adenylyl cyclase activity [8,21,22]. These data suggests that enhanced adenylyl cyclase activity is unlikely to be due to alterations in /33, subunits amount. However, confirmation of this requires immunological analysis with anti-/3 subunit antibodies.

The use of SG1 antisera in this study exclusively detects G~,il in lung membranes, since we were unable to detect any immunogenic polypeptides with the I3B, IC1 and IM10 antiserum [23]. Giu appears to be the favoured candidate G-protein responsible for inhibi- tion of adenylyl cyclase [24]. The studies of Kanaho et al. [25] confirm that the predominant Gi-like protein in lung is Gil ~. From these results, we speculate that the tonic inhibitory influence of G i upon adenylyl cyclase is mediated via G~II, and that this is the major pertussis- toxin-catalyzed ADP-ribosylation substrate. We cannot rule out the existence of a novel pertussis-toxin-sensi- tive G protein, although cDNAs have not been isolated for any other species as yet. Notwithstanding the im- munological levels of Gii I do not appear altered in hypersensitivity.

These results suggests that hypersensitivity is associ- ated with an up-regulation of/3-adrenoceptor response elements. The physiological consequence of this would be to allow higher concentrations of intracellular cyclic AMP to accumulate, thereby ensuring a new steady state level of active protein kinase A. This is of impor-

tance since/3-adrenoceptors agonists are inhibitory to the actions of certain inflammatory mediators in lung function, indicating that changes in sensitivity may re- flect adaptive changes in cell responses.

Acknowledgement

This study was supported by grants from the Na- tional Asthma Campaign, the Scottish Home and Health Department and the British Lung Foundation. We wish to thank Dr. I.W Rodger for helpful remarks concerning the establishment of the hypersensitivity model.

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