Modulation of octopamine-mediated production of cyclic AMP by phorbol-ester-sensitive protein kinase C in an insect cell line

324

Biochimica et BiophysicaActa, 970(1988)32J-332

Elsevier BBA12289

Modulation of octopamine.mediated production of cyclic A M P by phorbol-ester-sensitive protein kinase C in an insect cell line Gregory L. Orr*, John W.D. Gole**, Jyothi Gupta and Roger G.H. Downer Department of Biology, Universityof Waterloo, Waterloo(Canada)

(Received26April1988)

Keywords: eycficAMP;Octopamine;ProteinkinaseC; PhorbolEster;Adenylatecyclase;(Insee.,tcell) The presence of protein kinase C (EC 2.7.1.37) in an insect cell line has been demonstrated. Phorbol 12-myristate 13.acetate (PMA), in mieromolar concentrations, activated protein kinase C with a translocation of ~ enzyme from the cytosol to the paniculate fraction. Cyclic AMP production in the presence of PMA, octopamine and a combination of both increased in a dose.dependent and time-dependent fashion, The biologically inactive 4a-phorbol 12,13-dideeanoate had no effect on protein kinase C aelivity or on eeto~mine-mediated cydie AMP geduction, Pl'eb'ealment of the cells with pertnssis toxin had no elfect on the response of eeHs to oetopamine or PMA, However, pretreatment with cholera toxin resulted in increased eyelie AMP production which was further enhanced when both ehelera toxin and PMA were used in combination. Our data indicate that the oetopamine.mediated cyclic AMP la'oducfion is modulated by protein kinase C.

Introduction Octopamine, the monohydroxyphenoliz analogue of norepinephrine, occurs in high concentrations in nervous tissues and hemolymph of many invertebrates and in brain and sympathetically innervated tissues of vertebrates (reviewed in Refs. 1 and 2). In insects, octopamine functions as a

Presentaddresses: * Pestici:leResearchCentre,MichiganStateUniversity,East Lansing,~m,U,S.A. * * Departmentof Biology,CentralMissouriStateUnive~ity, Warrensberg,MO,U.S.A. Abbreviations: PMA, phorbol 12-myristate13-acct,-re; IP3, inositol trisphosphate; 1BlVIX,3-isobutyl-l-methylxanthin¢; PDD, 4a-phorbol-12,13-didecanoate;C, ~talytic subunit of adenylatecyclase. Correspondence: R.G.H. Downer, Department of Biology, Universityof Waterloo,Watedoo,Ontario,Canada,N2L 3G1.

neurotransmitter, neuromodulator and neurohormone [3] and has been propose,.4 as the sympathomimetie effector of physiological responses to excitation [4,5]. At least some of these physiological effects are mediated through interaction of the amine with a membrane receptor coupled to adenylate cydase (EC 4.6.1.1) [6-10]. Some hormones and neurotransmitters stimulate metabolism of membrane polyphosphoinositides to generate at least two intracellular second messengers: inositol trisphosphate (IP3) and di. acylglycerol (reviewed in Refs. 11 and 12). IP3 mobilises calcium from intracellular stores, whereas diacylglycerol stimulates a calciumactivated, phospholipid-d~pendent enzyme, prorein kinase C. Studies of blowfly salivary gland clearly demonstrate hormone-mediated hydrolysis of membrane polyphosphoinositides in insects [14-17] and preliminary studies in an insect cell llne indicate octopamine-mediated increases in intracellular calcium levels [18]. Protein kinase C has

01674889/88/$03.50©1988'~se~erSciencePublishersBN. (BiomedicalDivision)

325

been identified in insect neural tissue [19,29], but no relatonship has yet been established between ~topamine and the enzyme. A functional linkage between the adenylate cyclase system and inositol lipid hydrolysis has been demonstrated using turnout-promoting phorbol esters which bind to and activate protein kinase C [21-24]. Depending on the tissue studiO, phorbol esters can inhibit [25-27] or enhance [28-40] receptor-mediated cyclic AMP production. In the present study we demonstrate the presence of protein kinase C in an insect cell line and show, - to our knowledge, for the first time - its modulation of octopamine.sensitive adenylate cyclase. Materials and Methods

Cell cul;ure Cell lines of Choristo'leura fumi/erana (IBRICF1) were o;~tained from Dr. S, Sohi, Forest Pest Management Institute at Sault St. Marie, Ontario and reared in G-race's insect culture medium (pH 6.2) supplemented with 0.25~$ tryptose broth, 10~ fetal bovine s~rum and 0.00255 penicillin-streptomycin (5000 units and 5 mg/ml) in 75 cm2 flasks at 28°C. Cultures were initially seeded at 2.105 cells/ml and subcultured every 5-7 days as described previously [41]. Tissue preparation and incubation Intact cells. Whole cells were washed twice with physiological saline (168 mM NaCI/6 mM KCI/2 mM NaHCO3/6 mM NaH2PO4/2 mM CAC12/4 mM MgCI2/17 mM glucose (pH 6.2)) and resuspended in saline for assay of cyclic AMP production. The final assay volume was 100 #1 and contained tissue, test compound(s) or vehicle and 0.5 mM (IBMX). Unless otherwise stated, the cells were incubated for 30 rain at 30 °C in a shaking water-bath. Cell homogenates Whole cells ~,ere washed and resuspended in physiological saline containing PMA (6.5 ~tM) or vehicle (ethanol) and incubated at 30°C for 30 min in a shaking water-bath. Following incubation the cells were washed and resuspended in saline prior to homogenisation in a glass-Teflon homogeulser (30 strokes). The homo-

genate was used in a 10q/~i reaction m~ture containing tissue, test compound(s) and, where stated, 0.5 mM IBMX or a cyclic AMP spike (100 pmol). This solution was incubated for between 5 and 20 rain at 30 °C in a shaking water-bath. Crude membranes. Cells were washed twice in physiological saline, reconstituted into saline containing vehicle of PMA (6.5/~M) and incubated for 30 rain at 28°C. The cells were then washed with saline and homogenised in EDTA buffer (10 raM Tds/1 ram DL-dithiothreitol/1 mM EDTA (pH 7.0)) using a glass-Teflon homogeniser (30 strokes). Following centrifugation at 900 x g for 2 rain, the resulting supematant was centrifuged at 28000 × g for 10 rain and the pellet washed (28 000 x g, 10 min) with EGTA buffer (10 mM Tris/1 mM dithiothreitol/0.4 mM EGTA (pH 7.0)). The final pellet was reconstituted in EGTA buffer and incubated at 30°C for 20 mia in a final volume of 200 ~tl. The reaction mixture contained 75 mM Tris-acetate (pH 7.2), 0.05 mM GTP, 0.1 mM IBMX, 30 mM magnesium acetate, 1.5 mM ATP and the t~t compound(s) or vehicle. The reaction was initiated by the addition of ATE Membranes derived from cells treated with PMA received PMA as treatment.

Particulate and cytosolicfraction Cells were incubated with phorbol ester (6.5 /tM) or vehicle in a shaking water-bath for 1 h at 30 ° C. Following incubation, the cells were washed and resuspended in 7 nd ice-cold 25 mM Tris-HCl (pH 7.5)/0.25 M sucrose/2.5 mM MGC12/2.5 mM EGTA/50 mM 2-mercaptoethanol [19]. The cells were then homogenised and centrifuged for 5 rain at 300 × g with the resulting supernatant centrifuged for 1 h at 100000 x g to separate cytosolic and particulate fractions. The 100000 x g pellet was washed and resuspended in 7 ml homogenisation buffer with 0.35 Triton X-100, incubated for 1 h at 4°C and centrifuged at 100000 × g for I h to separate detergent-solubilised material. Protein kinase C activity present in both cytosolic and detergent-solubilised particulate fractions was measured. Toxin treatment Where indicated, cells were exposed prior to experimental preparation to pertussis toxin (500

326

ng/ml culturemedium) or choleratoxin(9 #g/ml culturemedium) at 28°C for 24 h or 3 h, respectively.

Assay of cyclic AMP All reactions were terminated by the addition of 1.0 ml 0.4 M perchloric acid. Following neutralisation to pH 6.0 with 2.5 M potassium bicarbonate and dilution with 0.5 M sodium acetate buffer, aliquots were assayed for cycfic AMP using a radioinL,nunoassay kit obtained from New England Nuclear (Lachine). Sample protein was determined using the Bio-Rad Protein Assay (BioRad Laboratories, Richmond, CA). Sample groups were compared using one-way analysis of variance [42]. In figures, S.E. for points was generally less than 10~ of the plotted value.

Assay of protein kinase C Protein kinase C was assayed by measuring the incorporation of 32p into histone from [y-32P]ATP using a slight modification of the method of Kishimoto et al. [43]. All reagents and buffers used in tissue preparation and protein kinase C determinations were prepared with Chelex-100treated H 2 0 (Sigma Chemical Co., St. Louis, MO) to remove Ca 2+ [44]. Briefly, the reaction mixture (0.25 ml) contained 5 ~tmol Tris-HCl (pH 7.5), 1.25/~mol MgCl2, 2.5 ~mol NaF, 50 pg historic and 2.5 nmol [~-32P]ATP((5-10)- 104 cpm/nmol). Where indicated, Ca 2+ (final concentration 0.5 mM) and phosphatidylserine (80 ~g/ml) were added to the reaction mixture. This preparation was incubated for 5 rain at 30 o C in a shaking waterbath and then a 25 ~1 aliquot was spotted at the origin (pretreated with 75 t~l 20% triehloroacetic acid) of a 1.5 × 13 cm chromatographic strip (Whatman, 31-ETCH.R). Ascending chromatography was performed as described by Huang and Robinson [45]. Subsequently, the filters were dried and approx. 3 cm, containing the precipitated histone, was placed in scintillation vials, 10 ml ScintiVerse E (Fisher Scientific) added and radioactivity counted in a scintillation counter. Basal activity was deducted from Ca2+/ phospholipid stimulated activity. The difference, indicative of protein kinase C activity, is expressed as pmoI PO4 transferred/rain per mg prote!.n.

Chemicals Octopamine, ATP, GTP, dithiothreitol, EGTA, IBMX, PMA, cholera toxin PDD, phosphatidylserine (bovine brain) and histone (type III-S) were purchased from Sigma, St. Louis, MO. Pertussis toxin and [y:2p]ATP (10-25 Ci/mmol) were obtained from ICN Biomedic~s (Canada), Montreal. PMA and PDD were dissolved in 100% ethanol (0.65 mM stock) and diluted prior to use. Vehicle controls were run in all experiments. Results

Untreated CF1 cells have 80~g of the protein kinase C activity in the eytosolic fraction and 20% associated with the particulate fraction (Table I). The activity of protein kinase C is enhanced by 63~ in cells incubated in the presence of 6.5 ~tM PMA; moreover, PMA treatment shifts the majority (92~) of protein kinase C activity to the particulate fraction. The biologically inactive phorbol ester, PDD (6.5 #M) had no effect on protein kinase C activity or distribution. Initial experiments using intact CF1 cells showed PMA to enhance basal cyclic AMP production in a dose-dependent fashion, with a 4.5-fold increase observed at the highest dose tested (6.5/tM, Fig. la). The response to 0.1 mM octopamine was enhanced in a similar fashion (Fig. la), with the peak effect (1.9-fold) occurring between 1.3 and 6.5 /tM PMA. The absolute increase in cyclic AMP, obtained by subtracting the basal from the octopamine~induced level at different concentrations of PMA indicates significant stimulation at

TABLE I PROTEIN KINASE C ACTIVITY IN CF1 CELLS PRETREATED WITH PMA OR PDD The values presented are the Ca2+-stimulaled activity in the presence of phospholipid (phosphatidylserine) corrected for basal activity. Specific activity is expressed as pmo132p incorporated/rain per rag protein. Values are mean + S.E. for four determinations. Assays were performed in duplicate.

Control PMA (6.5 aM) PDD (6.5/~M)

Cytosol

Particulate

74.82+9.14 11.70±0.70 63.93 :~5.68

19.21+ 1.56 141.4 +22.52 22.79+ 3.25

327

1400

E 0

1ooo

E

.j"

/'

1~00

1000

/+octopnmlne

c

/

a 900

£E

800

~~

800

o

o

E

E& 600

•/ ' / "

u 400,

L

o~O

700

6oo[

U 200

.~=~

~

- Octooomine

50040

!.....~/I'~

I [

010.8

10-7

[PMA] (M)

10-6

~PMA]

iO-S

(M)

Fig. 1, Effect of PMA on cyclic AMP production in intact CFI cells. (a) PMA dose response for cyclic AMP accumulation in the presence and absence of 0.1 mM octopamin¢; (b) enhancement of octopaminc-mediated cyclic AMP production by PMA. Points calculated from data in (a), Each point represents the mean of five determinations.

10 nM phorbol ester and that the ECho for PMA is approx. 0.3/tM (Fig. lb). Cyclic AMP production in the presence of PMA (6.5 laM), octopamine (0.1 raM) and a combination of these treatments increased in a time-dependent fashion for at least 30 rain (Fig. 2) with the greatest production seen in the presence of both octopamine and PMA. PMA enhances octopamine-mediated cyclic AMP production over that of control throughout the time period studies.

The synergistic effects of PMA (6.5/~M) are seen at most of the stimulatory concentrations of octopamine (1.0-3000/tM) (Fig. 3). The K~ for octopamine is difficult to determine in these cells as peak cyclic AMP production does not occur at doses as high as 10 ram octopamine. However, in the presence of PMA, maximal production was reached at 1.0 mM octopamine and the Ka was determined to be 0.013 raM. The biologically inactive phorbol ester, PDD (6.5 FM), had no effect on basal or octopamine-mediated cyclic AMP production (Table II). In cell homogenates, the degradation of added cyclic AMP is unaffected by pretreatment of the cells with PMA (6.5 pM, 30 rain) with complete degradation occurring at 20 ~ (Fig. 4). Endoge-

/

18oo 14oo

•~to~gQ~lr'te+ PMA ";

$ 1000

~--

aoo

- 600 ~< .~

u

400

2oo o

~,_..~, -0

5

10

15

- =-;E," 20

25

30

Time, (re=n)

Fig. 2. Time.course of cyclic AMP production in intact CF1 cells. Cells were incubated in the presence of vehicle, PMA (6.5 FM), octopamine (0.1 mM) or a combination of these doses of PMA and octopamine for the times designated. Each point represents the mean of five determinations.

328 100

;/;------"

t800

I~ | /

+PMA

/

1600 1400

60

c

1200

4o

c

lOOO,

c ,_

2c

i 800

0 2 4 6 8 10 12 14 16 18 20 Time (ram)

600

Fig. 4. Effect of P]VL~on cyclic AMP degradation in CF1 ceil homogenates, Following incubation with PMA (6,5/tM) (e) or vehicle (11)cells were homogenized and aliquots cou'raining 100 pmol added cyclic AMP were incubated for the designated timv. Any remaining cyclic AMP was then determined. Values equal the mean of five determinations,

400{ u

U

OJ t

10-0

10-7

= 10-e

t0-5

10.4

10-3

10-2

I'octopomine] (M~

3. Effect of PMA on the dose-dependent increase in cyclic AMP mediated by octopamine in intact CF1 cells. Ceils were incubated with the designated octopamine concentration in the presence or absence of PMA (6.5 tiM). Each point represents the mean of 5-15 determinations from three separate experi. ments. *, significantly different from values in the absence of PMA, P < 0,05. Fig.

nous cyclic AMP was also not detectable after 20 rain, but in the presence of the phosphodiesterase inhibitor, IBkOC (0.5 mM), homogenates of PMA-pretreated cells show an enhanced (5.95fold) production of cyclic AMP over that seen in control cells (Table III). In control and PMA-preTABLE II

treated cells the inclusion of IBMX and an exogenous cyclic AMP spike resulted in the complete recovery of the spike and endogenous cyclic AMP after 20 min incubation. The importance of an hmibitory guanine nucleotide-binding subunit (Ni) was evaluated by pretreating the cells with pertussis toxin (500 ng/ml, 24 h) and then measuring the cyclic AMP TABLE I!I EFFECT OF PMA PRETREATMENT ON CYCLIC AMP DEGRADATION IN HOMOGENATES OF CF~ CELLS Zero time cyclic AMP without spike is not detectal~le. Zero time cyclic AMP with spike, + PMA = 561.66+ 26.42, -PMA =617.79+34.32, Values are mean+S.E for five determinations, n.d., no cyclic AMP detected. Treatment

EFFECT OF PDD ON CYCLIC AMP PRODUCflON IN INTACT CFI CELLS Values are mean ± S.E. for five determinations. Treatment Control PDD (6.5 pM) Octopamine (03 raM) Octopamine (0.1 mM) + PDD (6.5 t t M )

•

CyclicA M P production (pmol/30 min per mg protein) 120.24+ 18.84 157.39+ 28.18 976.3! + 72.35 1097.56-1-106.89

mMX (o.5 raM)

Cyclic AMP increase over zero time (pmol/20 rain per mg protein)

control

PMA (6.5 ~M)

n.d. 49.73 + 8.53

n.d. 296.02± 20.39

Spike (100 pmol cyclic AMP/sample) n.d. n.d, Spike (100 pmol cyclic AMP/sample) + IBMX(0.5raM) 776.61+62.63 * 922.34±32.01 * * Not significandy different from IBMX controls plus spike values, P > 0.05.

329 TABLE IV

TABLE V

EFFECT OF PERTUSSIS TOXIN PRETREATMENT ON CYCLIC AMP PRODUCTION IN INTACTCF! CELLS

EFFECT OF PMA AND CHOLERA TOXIN ON CYCLIC AMP PRODUCTION IN. INTACT CFt CELLS

The treatment described followed 24 h preincubation with pcrtussis toxin (500 ns/ml) or vehicle.Control withoutpertussis toxin (-PT'.)=112.47+6.57, with pertussis toxin (+PT)= 87.89+_7.44, significantlydifferent, P < 0.05; treatments with pertussis toxin vs. without pertussis toxin, not significantly different, P > 0.05. Valuesare mean+ S.E. for fivedetermina. tions.

Control=248.19±35.67 pmol cyclic AMP/30 min per mg protein. Values are mean±S.E, for five determinations.

Treatment

Increase in cyclicAMP production (pmol/30 rain per mg protein) -PT

+PT

Octopamine(03 mM) 858.15± 49.86 745.08+ 32.19 PMA(6.SgM) 465.70+ 62.59 465.01+ 14.51 Octopamine(0.1 raM) +PMA (6.5 ttM) 2575.16:1:152.90 2174.04+107.67

production after washing. This pretreatment resulted in a slight, but significant, reduction in basal cyclic AMP production, although the response to octopamlne (0.1 mM), PMA (6.5/~M) or a combination of octopamine and PMA was unaffected (Table IV). A similar result was observed following a 4 h pretreatment with pertussis toxin (unpublished obsemtdons). The role of the stimulatory guanine-nucteotide binding subunit (Ns) was investigated using

Treatment

CyeticAMP increase Theoretical (pmol/30 rnin increaseif per mg protein) fullyadditiw:

PMA (6.5 ~M) 432.27:1-28.36 Cholera toxin (9 pg/ml) 1505.88+ 122.27 Choleratoxin (9 ~glml) +PMA(6.5/~M) 15244.31±760.7574 193835 -

cholera toxin (Table V). Following a 3 h pretreatment of the cells with PMA (6.5 ~M) or cholera toxin (9 #g/ml) cyclic AMP production increased by 1.7- and 6.l-fold, res~.xtively. Cyclic AMP production following pretreatment with b o b com. pounds was 7.9-fold greater than would be ex. pected if the effects of the two compounds were totally additive. The effects of PMA on adenylate cyclase activity were further investigated in crude membrane preparations using compounds known to activate adenylate cyclase via specific routes (Table VI). In

'fABLE VI EFFECT OF PMA ON CYCLICAMP PRODUCTION IN CRUDE MEMBRANEPPd~PARATIONSOF CF1 CELLS Valuesof increaseover control werecalcula.,:dagainst the controlvaluesfor each day's experiment.All valuesare the mean± $.E. for 5-15 det~minafions.Values ,Y0talnedfrom three separate experiments. Treatment

CyclicAMP (pmol/minper mg protein)

Increaseover control (pmol cyclicAMP/ rain per mg protein)

-PMA Control N~F (1 raM) GppfNH]p(10 ~tM) Mn2+ (30 mid)

574.15+ 49.76 847.99± 74.09 * 2 811.80+ 114.46 * 638.17+ 23.45

191.86± 70.23 2426.81± 114.60 -

+ PMA (6.5 ~,M) Control NaF(1 mM) GpplNH]p(lOpM) Mn2+ (30raM)

754.81± 52.77 * 1262,99+ 74.09 * 3470.33i 97.31 * 706.55+ 46.83

195.19± 24.74 420.86+ 42.30 ** 2855.06+ 97.27 ** =

* Values significantlydifferentfrom controh, P < 0.05. Values sisnificantJydifferent from values obtained in the absenceof PMA, P < 0.02.

**

330

tiffs system, PMA (6.5/~M) significantly increased (31~) basal cyclic AMP production. Sodium fluoride (1 raM) increased cyclic AMP production in the presence or absence of PMA (6.5/~M), but the response was enhanced 2.2-fold when PMA was included in the reaction media. A similar, but smaller (18%), enhancement was observed in the presence of the nonhydrolysable GTP analogue, Gpp[NH]p (10/~M). In contrast, manganese ion (30 raM), in the absence of added Mg2+, had no significant effect on cyclic AMP production in the presence or absence of PMA. Discussion

The present study confirms earlier reports of protein kinase C in insect tissues [19,20]. The ability of phorbol ester to enhance protein kinase C activity in CF1 cells is consistent with studies using synaptosomal preparations of locust ganglia which showed enhanced incorporation of 32po4 into historic in the presence of Ca 2 *, phosphatidylserine and phorboI ester [20]. However, basal incorporation with Ca2+ and phosphatidylserine in the absence of phorbol ester was not reported making comparison with the present study difficult. The phorbol ester-mediated shift of protein kinase C from the cytosolic to the particulate fraction in CF: cells has not previously been shown in insects but has been reported in vertebrate preparations [46-48]. The response of protein kAnase C in CFt cells to PMA and the lack of effect of the biologically inactive phorbol ester, PDD [47], indicate a basic functional similarity between insect and vertebrate protein kinase C. The presence of protein kinase C in CF~ cells suggests that diacylglyeerol, produced as a result of membrane phosphatidylinositol metabolism, may be acting as a second messenger in these cell~. A possible physiological role of d~acylglycerolactivated protein kinase C involves the modulation of hormone-sensitive adenylate cyclase by phosphorylation reactions [40,49]. We studied this relationship in CF1 cells by monitoring the effects of PMA on octopamine.sensitive adenylate cydase under conditions which would facilitate the activation of protein kinase C. PMA, presumably acting through protein kinase C, enhances basal and octopamine-mcdiated cyclic

AMP production in a dose- and time-dependent fashion. The involvement of protein kinase C in this effect is further suggested by the lack of enhancement seen in the presence of PDD which does not activate the enzyme (Table I; [47]). These observations are consistent with those seen in a number of vertebrate preparations (see Int, oOuction) and are the first reported in insect cells. Moreover, this study adds another level of complexity to the control of octopamine-mediated events by indicating that octopamine-sensitive adenylate cyclase can be modulated by agents stimulating the metabolism of membrane phosphoinositides leading to the activation of prorein kinase C. It is possible that octopamine itself may stimulate the production of IP3 and diacylglycerol and, although this has not yet been desc;dbed, octopamine effects that are not mediated by cyclic AMP have been reported [50] and octopamine-mediated increases in intracellular calcium levels have been described in an insect cell line [18]. There are several sites associated with the production and degradation of c'ycfic AMP which could serve as a substrate(s) for PMA-activated protein kinase C. Cyclic AMP phosphodiesterase activity increases [51] and decreases [52] in response to phorbol ester. In the present study, cyclic AMP degradation is unaffected in homogenates of cells treated with PMA, indicating no attenuation of phosphodiesterase activity. Furthermore, in tissue homogenates, the phosphodiesterase inhibitor IBMX totally inhibits cyclic AMP degradation (as determined by spike recovery) and basal cyclic AMP production is enhanced by PMA. Therefore, in CF1 cells it seems that PMA is acting at a site other than phosphodiesterase to enhance cyclic AMP production. Protein kinase C phosphorylates the a i subunit of N i in platelet membranes [49,53] reducing its ability to inhibit adenylate cyclase activity. This mechanism is thought to explain the ability of phorbol ester to enhance adenylate cyclase activity in platelet membranes and $49 lymphoma cells [34]. However, in intact platelets, sites other than N i are involved with phorbol ester-mediated modulation of adenylate cyclase activity [27]. Furthermore, purified catalytic subunit (C) of bovine caudate adenylate cyclase is a substrate for pro-

331

tein kinase C [54] and phorbol ester treatment of frog erythrocytes results in phosphorylation of C [40]. Therefore, a variety of potential sites associated with adenylate cyclase subunits may be involved in the modulation of this enzyme by

phorbol ester-activated protein kinase C. In $49 lymphoma cells, pertussis toxin, which inhibits Ni t~rectly [55], and phorbol ester enhance agonist-mediated cyclic AMP production, but their efiects are not additive, indicating a common mode of action through N i [34]. In CF1 cells, pertussis toxin does not enhance basal or octopamine-mediated cyclic AMP production nor does it interfere with the effects of PMA. This observation suggests that CFi cells do not contain an Ni subunit, do not have a pertussis toxin-sensitive Nt and/or oetopamine-sensitive adenylate cyclase in these cells is not re,dated by an N i subunit. Therefore, if PMA is acting through an inhibitory subtmit it would appear that it is not pertussis toxin.sensitive. This result may or may not be t~pic~ of insect tissue but it is relevant to note that agonist- or guanine~nucleotide-mediated inhibition of adenylate cyclase has not been re-

ported in insects. Cholera toxin, which directly activates the stimulatory guanine.nucleotide-binding protein (Ns) [56], greatly enhances cyclic AMP production in CF1 cells, confirming earli.er evidence indicating the presence of N~ in these cells [41]. As previously observed in $49 lymphoma cells [281 the response to cholera toxin is greatly enhanced in the presence of PMA, suggesting facilitation of the interaction of as and C. Similar results were seen in membrane preparations, where PMA enhanced the effects of compounds which permanently activate Ns (NaF, Gpp[NH]p). Clearly, in CFI cells, PMA enhances the effects of compounds acting via Ng, but whether this is due to protein kinase C-mediated phosphorylation of N s, C, an inhibitory subunit or some other membrane component is tmclear. The possibility of C beins a substra~c for protein kinase C has been dL~.ussed above. The adenylate cyclase of CF1 ceils is umque in that it does not respond to forskolln [41] but does produce cyclic AMP in the presence of Mn2+ (in the absence of Mg 2÷) which is known to act dlrecdy on C [57]. In membrane preparations of CF1 cells

PMA does not ~ter the effects of Mn2+, indicating that C is not modified by PMA treatment. Future studies will determine the substrates for protein kinase C in CF1 cells and help determine the mechanism by which prot~a kinas¢ C modulates octopamine-sensitive adenylate cyclase. Acknowledgements Supported by grants from Natural Sciences and Engineering Research Cnuncil of Canada and Agricultural Research Division, Americaa Cyanamid.

References 1 Robertson, H.A. (1981) in Neurochemis~ryand Neuropharmacology,Vol. 5 (Youdima,M.B.H., Lovenberg,W., Shanmn, D.F. and Lagnado,J.R., eds.), ~: 47-73, John Wileyand Sons,N~v York. 2 David,J.C. and Coulon,J.E (1985)Prog. Neurobiol.24, 141-185. 3 Orchard,1. (1982)Can.J. 7.ool.60, 659-669. 4 Hoyle,G. (1975)J. Exp.7_,ool.193,425--43I. 5 Downer,R,G.H.(1979)J. InsectPhysiol.29, 55-63 6 Nathanson,JA. Greangard,P. (1973)Science180,308-310. 7 Gole,J.W.D. and Downer,R.G.H.(1979)Comp.Biochem. Physiol.64C,223-226. 8 Orchard, I., Gole, J.W,D. and Downer, R.G.H. (1983) Brain Res. 228,349-353. 9 Downer,R.G.H.,On', G.L, Gole,J.W.D. and Orchard,I. (1984)J. InsectPhysiol.30, 457-462. 10 Evans,P.D. (1984)J. Physiol.348,30?-324. 11 Ikrfidge,MJ. (1984)Biochem.J. 220, 345-360. 12 Berridge,MJ. (1987)Annu. Rev,Biochem.56,159-193. 13 AMel-Latif,AA. (1986)Pharmacol.Rev.38, 227-272. 14 Berridge,MJ. (1~3) Bioehem.J. 212,849-858 15 Lito~h, I., Lee,H.S.and Fain,J.N. (1984)Am. J. Physiol. 246, C141-C147. 16 Sadler, K., Litosch,L and Fain, J.N. (1984)Biochem.J. 222, 327-334. 17 Litosch,I., Wallis,C and Fain,J.N. (1985)J. Biol.Chem. 260, 5464-5471. 18 Jahagirdar,A.P., Milton,G., Viswanatha,T. and Downer, R,G.H. (1987)FEBSl.ett. 219(1),83-87. 19 Kuo, J.F., Anderson,R.G.G., Wise, B.C., Mac~etlove~I , ~lomonsson,J., Brackett,N.L, Katoh,N., Shoji,M. and Wrerm, R.W. (1980) Proc. Natl. Acad. Sci, USA 77, 7039-7043. 20 Ktfipper, M. and Breer, H. (1987) Neurocho'u. int. 10; 323-328. 21 Castagna,M., Takai,Y., Kaibuchi,K., Sano,K., Kikkawa, U, and Nishizulm,Y. (1982)J. Biol.Chem.257,7847-7851. 22 Niedel,J.E., Kulm,LJ. and Vandenburk,G.R.(1983)Proc. Natl. Acad.Sci.USA 80, 36-40.

332 23 Kikkawa, U., Takai, Y., Taaaka, I"., Miyake, R. and Mishizuka, Y. (1983) J. Biol. Chem. 258, 11442-11445. 24 I.,¢¢, M.H. and Bell, R.M. (1986) J. Biol. Chem. 261, 14867-14870. 25 Sibley, D.R., Nambi, P., Peters, J.1L and Lefkowitz, RJ. (1984) Biochem.Biophys. Res. Conunun. 121, 973-979. 26 Meurs, H., Kauffman, H.F., Timmermans, A., Van Amsterdam, F.Th.M., Koeter, G.H. and De Vries, K. (1986) Biochem. Pharmacol. 35: 4217-4222. 27 Williams, K,A., Murphy, W. and Haslam, ILL (1987) Biochem. J. 243, 667-668. 28 Bell, J.D., Buxton, I.L.O. and Brunton, L.L (1985) J. Biol. Chem. 260, 2625-2628. 29 Hollingsworth,E.B., Sears, E.B. and Daly, J.W. (1985) FEBS Lctt.184,339-342. 30 Nabika, T., Nara, Y., Yamori, Y., Lovenbcrg, W. and End<), J. (1985) Biochem. Biophys. Res. Commun. 131, 30-36. 31 Cro~'~,M.J. and Canonico,P.L.(1985)Biochem. Biophys. Res. Commun. 129,404-410. 32 Sugden, D., Vanacak, J., Klein, D.C., Thomas, T.P. and Anderson, W.B. (1985)Nature 314,359-361. 33 Jakobs, K.H., Bauer, S. nd Walan~-be,Y. (1985) Eur. J. Biochem. 151,425-430. 34 Bell,£D. and Brunton, LL (1986) J. Biol.Ch~:m. 261, 12036-12041. 35 Olianis,M.C. and Onali, P. (1986) J. Neurochem. 47, 890-897. 36 Karbon, E.W., Shenolikar,S. and Enna, S.J. (1986) J. Neurochem. 47,1566-1575. 37 Pines,M., Santora,A. and Spiegal,A. (1986) Biochem. Pharmacol.35, 3639-3641. 38 Summers, S.T.nd Cronin, MJ. (1986)Biochem. Biophys. Res. Commun. 135,276-281. 39 Abou-Samra, A., Harwood, J.P.,Manganic¢llo,V.C.,Cart, KJ. and Aguilera,G. (1987)J.Biol.Chem. 262,1129-1136.

40 Yoshimasa,T., Sibley, D.R., Bouvier, M., Lefkowitz, R.J. and Caron, M.G. (1987) Nature 327, 67-70. 41 Gole, J.W.D., On', G.L. and Downer, R.G.H. (1987) Biochem. Biophys. Res. Commun. 145,1192-1197. 42 Zar, J.H. (1974) Statistical Analysis, Prentice.Hall, t~n. glewoex:lCliffs. 43 Kishimoto, A, i'akai, Y., Mori, %, Kakawa, U. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276. 44 T.S. and Wang, J.H. (1973) J. Biol. Chem. 248(17), 5950-5955. 45 Huang, K. and Robinson, J.C. (1976)Anal. Biochem. 72, 593-599. 46 Kraft, A.S. and Anderson, W.B. (1983) Nature 301, 621-623. 47 Kilo, J.F., Shoji, M., Girard, P.R., Maze:., GJ., Turner, R.S. and Su, H. (1985) Adv. Enzyme Regul. 25, 387-400. 48 Gopalkrishna, IL, Barsky, S.H., Thomas, ILP. and Anderson, W.B. (1986) J. Biol. Chem. 261,16438-16445. 49 Jakobs, K.H., Watanabe, Y. and Bayer, S. (1986) J. Cardiovas¢. Phatma¢ol. 8 ([;uppl. 8), $61-$64. 50 Evans, P.D. (1984b) J. Physiol. 348, 325-340. 51 Solomon, S.S. and Palazzolo, M. (I986) Am. J. Med. Sci. 292,182-1M. 52 Irviae, F., l~/ne, NJ. and Houslay, M.D. (1986) FEBS I~tt. 208, 455-459. 53 Katada, %, C,-ilman, A.G., Wamal~, Y., Bauer, S. and Jakobs, K.H. (1985) Eur. J. Biochm. 151, 431-437. 54 Yoshimasa, T., Bourier,M., Bcnovic, J.L.,Amlalky, N., l,¢fkowitz,RJ. and Caron, M.G. (1986) Clin. Rcs. 34, 689A. 55 Ui, M. (1984) Trends Pharm. Sci. 5, 277-279. 56 Sp;.egal,A.M. (1987) Mol. Cell. EndocrinoL 49,1-16. 57 Cech, SA'., Broaddus, W.C. and Maguire, M.E. (1980) Mol. Cell. Bieehem. 33, 67-92.