Archs oral Biol. Vol. 35, No. 11,pp.885-890, Printed in Great Britain. All rights reserved
1990 Copyright
0
0403-9969/90 $3.00 + 0.00 1990 Pergamon Press plc
EFFECT OF AGEING ON ADENYLATE CYCLASE ACTIVITY AND G-PROTEINS IN RAT SUBMANDIBULAR SALIVARY GLANDS* S. N. AHMAD,
S. Q. ALAM? and B. S. ALAM
Louisiana State University Medical Center, Department of Biochemistry and Molecular Biology, New Orleans, LA 70119, U.S.A. (Accepted 23 May 1990)
Summary-Membranes were prepared from the submandibular salivary glands of male young (3-monthsold) and aged (24-months-old), Fisher 344 rats, and assayed for adenylate cyclase activity and for the ribosylation of G-proteins in the presence of [32P]-NAD+ and cholera toxin. Adenylate cyclase activity (basal, fluoride-, forskolin-, and isoproterenol-stimulated) was significantly lower (p < 0.01) in the glands of aged rats. The pattern of ribosylation was different between the 2 groups. Two cholera toxin-specific bands (M, 42,000 and 44,000) were ribosylated in the glands of young rats whereas in aged rats, only an M, 42,000 band was clearly observed. The incorporation of 32P from NAD+ into the M, 42,000 and 44,000 bands increased with the time of ribosylation (7.5-90 min). In aged rats, however, the incorporation of 32P into the M, 42,000 band was much slower and did not increase much after 60 min of ribosylation. At each time point, the extent of ribosylation was lower in the membranes from the aged rats. These findings suggest that the decrease in adenylate cyclase activity in the submandibular salivary glands of aged rats may be due partly to the changes in their levels of G, as a result of ageing. Key words: salivary gland, adenylate cyclase, G-protein.
INTRODUCTION
The adenylate cyclase system consists of the receptor, G-proteins (stimulatory, G, and inhibitory, Gi) and the enzyme adenylate cyclase. There is evidence that some receptor systems which act through G-proteins are impaired in ageing @‘Conner, Scarpace and Abrass, 1983; Baker et al., 1985; Narayanan and Tucker, 1986; Feldman et al., 1984). Deficits in these systems may involve #changes in receptor number, the G-proteins, or the catalytic subunit of adenylate cyclase. An impaired stimulation of adenylate cyclase with age in rat heart was reported to result from a decrease in the amount of G, and not from a loss of B-adrenergic receptors @‘Conner et al., 1983). Similarly, in aged rat heart, the density of muscarinic cholinergic receptors was not changed, but these receptors formed fewer high-affinity binding complexes with Gi (Baker et al., 1985). Decreased coupling of the receptor to Gi has been shown to result in impaired muscarinic agonist inhibition of myocardial, p-adrenergic: receptor-stimulated, adenylate cyclase activity (Narayanan and Tucker, 1986). In leucocytes from aged humans, the density of /I-adrenergic receptors is not changed, but receptorstimulated adenylate cyclase activity is decreased because of impaired coupling between the receptor *Presented in part at the International Association for Dental Research/American
Association for
Dental
Research,
Cincinnati, Ohio, 7-l 1 March 1990. tTo whom correspondence should be addressed. Abbrezktions: G-protein, guanine nucleotide-binding regulatory protein; SDS-PAGE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis.
and G-proteins (Feldman et al., 1984). Such impaired coupling with age may result from an age-related loss of G-proteins which prevents formation of the high-affinity binding complex. In several systems, such as rat lymphocyte (Abrass and Scarpace, 1982), heart @‘Conner et al., 1983), erythrocytes (Farfel and Cohen, 1984), lung (Scarpace and Abrass, 1983), and guinea pig ureter (Wheeler et al., 1986), a significant decrease in the ability of forskolin to stimulate adenylate cyclase activity has been observed with ageing. As forskolin activates adenylate cyclase by direct binding to the catalytic unit of the enzyme (Clark et al., 1982; Seamon, Padgett and Daly, 1981; Seamon and Daly, 198la), these studies suggest age-related changes in that catalytic unit. Several aspects of stimulus-secretion coupling in rat parotid acinar cells during ageing have been characterized (Ito, Baum and Roth, 1981; Ito et al., 1982; Bodner et al., 1983; Gee et al., 1986). The /I-adrenoreceptor signal-transduction coupling is unchanged with age, with respect to exocrine protein secretion in rat parotid gland (Ito et al., 1981). However, Schmid et al. (1988) found that the response of isoproterenol, as measured by CAMP production and adenylate cyclase activity in parotid gland homogenates, was reduced in older (16-18 weeks) as compared to younger (6-8 weeks) rats. Kousvelari et al. (1988) have reported a decrease in the incorporation of [‘HI-mannose into N-linked glycoproteins in parotid acinar cells of aged rats. After ,!I-adrenergic activation, however, cells from both young adult and aged glands showed increased N-linked protein glycosylation to almost the same extent. 885
S. N.
886
hiMAD
To our knowledge there are no published reports of the effects of ageing on the fi-adrenergic-adenylate cyclase complex in the submandibular salivary gland. We have now investigated adenylate cyclase activity and the G-proteins in these glands of young and aged rats. MATERIALS AND METHODS
Male Fisher 344 rats were purchased from the National Institute on Aging (Bethesda, MD). Young rats were 3-months-old; the aged rats were 24months-old. According to the information provided by the National Institute, the rats had been tested for viral and mycoplasmal antibodies. No clinical or histopathological evidence of infectious illness was detected. The rats had been fed NIH 31, TEKLAD diet while housed at the Institute. Upon arrival in our laboratory, they were housed individually in wirebottomed cages and fed a standard laboratory diet (Purina rat chow) for about 1 week until they were killed. The body weight of the young rats was 190-275 g, and that of aged rats, 380490 g. After killing, the submandibmar salivary glands were dissected out, cleaned from the sublingual glands, and then crude membranes were prepared from them, as described previously (Ludford and Talamo, 1983; Ahmad, Alam and Alam, 1990). In brief, the glands were homogenized in 10 volumes (w/v) of ice-cold 10 mM tris-HCl, pH 7.4, containing 0.3 M sucrose and 0.5 mM EDTA in a Potter-Elvejhem glass homogenizer with 20-26 up and down strokes at 4°C and 3000 rpm; the resultant homogenate was then strained through two layers of cheese cloth and centrifuged for 10 min at 2000g. The pellet was washed twice by suspending it in 10 volumes of sucrose-tris medium and centrifuging (as above), and then by resuspension and centrifugation in 10 volumes of 50 mM tris and 0.05 mM EDTA, pH 7.4. The final pellet was suspended in sucrose-tris medium at a protein concentration of 5-10 mg/ml and stored in small portions in liquid nitrogen until analysis. For use, thawed membranes were washed once with 10 mM tris-HCI, pH 7.4, and the pellet resuspended in 5 volumes (per g wet wt) of 50mM potassium phosphate buffer, pH 7.3. Protein concentration in the membranes was measured in replicates of samples by Bradford’s method (1976) after solubilizing the membranes with NaOH as described by Kawai and Arinze (1983), using bovine serum albumin as standard. ADP-ribosylation of membrane proteins
ADP-ribosylation of membrane proteins was done with GI-[~~P]-NAD+ and cholera toxin, as described by Johnson et al. (1978) but with some modification.
et
al.
Cholera toxin was preactivated by incubation of 1 mg/ml protein for 10 min at 32°C in a medium containing 10mM tris, 50mM glycine, pH 8.0, 20 mM dithiothreitol, 50 mM NaCl, 0.2 mM EDTA and 0.6 mM sodium azide. Activated toxin was diluted into the final incubation mixture. The final labelling mixture (100 ~1) contained 50 mM potassium phosphate buffer, pH 7.3, 2OOpg membrane proteins, 10 mM thymidine, 20 mM arginine-free base, 5 mM ADP-ribose, 100 PM GTP, 5-10 FM cc-[32P]-NADC (sp. act. 25 Ci/mmol), 1Opg of toxin or toxin buffer. The reaction mixture was incubated for (r90min at 32°C. Blanks (membrane without cholera toxin) were incubated for 90min. Reaction was stopped at different times by adding 1 ml of ice-cold, 50 mM potassium phosphate buffer, pH 7.3, containing 10mM thymidine, 20mM arginine and 5 mM ADP-ribose. Tubes were kept on ice-cold water, vortexed and centrifuged at 20,OOOg for 30 min. All the supernatant was removed without disrupting the pellet. The pellet was resuspended in 60 ~1 of 1 mM HEPES, pH 8.0, containing 100pM MgCl,, 100pM EDTA and 0.7% lubrol PX. The membranes were dispersed with a Pasteur pipette and solubilized by allowing them to stand on ice with occasional vortexing. The samples were then centrifuged at 20,OOOg for 2 h and ADPribosylated proteins were examined in the supernatant. Eiectrophoresis
SDS-PAGE was carried out as described by Laemmli (1970) in 1.5 mm-thick slab gel using 10% acrylamide. Lubrol extract containing 60 pg protein was loaded on the gel and electrophoresed at a constant 70 V in the stacking and 200 V in the sieving gel. Gels were stained for 2 h in 0.1% Coomassie blue R-250, prepared in destaining solution (7% acetic acid and 10% methanol), destained, and dried under vacuum. Dried gels were exposed for 72 h to Kodak X-omat film for autoradiography. The intensity of 3zP incorporation was monitored on an image-array processor. Low molecular-weight standards were: bovine serum albumin, hen egg white ovalbumin, bovine carbonic anhydrase and soybean trypsin inhibitor. Adenylate cyclase assay
Adenylate cyclase activity was measured essentially according to the method of Salomon et al. (1974) by sequential chromatography on columns of Dowex cation-exchange resin and alumina to isolate [32P]-cAMP formed from a-[‘*PI-ATP by adenylate cyclase. Details of the procedure have been described by Alam and Alam (1986). In our study, 0.5 mM ATP was used as substrate. The recovery of reaction
Plate 1 Figs 1. and 2. Autoradiograms of lubrol extracts of [‘*PI-ADP-ribosylated membranes from submandibular salivary glands of young (Fig. 1) and aged (Fig. 2) rats. Membranes were incubated with [‘*PI-NAD+ in the absence (lane 1) or presence of cholera toxin (lanes 2-8) for different time periods and lubrol extracts were prepared. Proteins were separated on 10% acrylamide gel by SDS-PAGE and the gels were exposed to Kodak X-Omat film for 72 h. Molecular weight of marker proteins are shown to the left of the figure. Arrows indicate cholera toxin-specific changes in proteins M, 42,000 and 44,000 in young rats (Fig. 1) and M, 42,000 in aged rats (Fig. 2).
887
Ageing and salivary gland adenylate cyclase
7
a
t
+
+
45
60
75
90
1
2
-
t
-t
+
+
90
7.5
15
30
3
4
5
6
42.?-
Cholera Time
Toxin (min)
Fig. 1
1
2
3
4
5
6
7
8
-
+
+
t
t
t
t
•k
90
7.5
15
30
45
60
75
SO
42.7
31 .o
21.5
Cliolera Time
Toxin (min)
Fig. 2
Plate
I
888
S. N. AHMAD Table
I. Adenylate cyclase activity in submandibular (24-months-old)
Type rats
of
Young Aged
et al.
salivary glands of young (3-months-old) Fisher 344 rats
Basal -Mn’+
Basal +Mn*+
+ Fluoride*
10.5 * 0.3 4.6 k 0.5t
50.0 + 2.4 14.9 k 5.2f
558 f 26.8 174+ 11.ot
+ Forskolin* 350 * 19.4 89.5 & 3.5t
and aged
+ Isoproterenol’ 15Ok4.4 48.7 f 1.51
Values are mean + SEM of 3 separate assays, each done in triplicate. The enzyme activity is shown as pmol of cAMP/mg proteimmin. *The stimulated activity in the presence of fluoride (15 mM), forskolin (0.1 mM) or isoproterenol (20 PM) was measured in the presence of 10 mM MnCl,. Values with superscripts are significantly different from the corresponding values in young rats (Student’s t-test: tp < 0.001; QJ < 0.01).
product was monitored [3H]-cAMP.
by adding internal
standard,
RESULTS
Adenylate cyclase activity in the membranes from young and aged rats is shown in Table 1. The enzyme activity (basal, fluoride-, forskolin-, and isoproterenolstimulated) was significantly lower (p < 0.01 in most cases) in the membranes of aged rats. The non-stimulated (basal) activity in such membranes was 44% (- Mn*+ ) and 30% (+ Mn*+ ) of that in young rats. Similarly, the stimulated activities in the membranes of aged rats were 2633% of the corresponding activities in those of the young rats. The time-course for ribosylation of the membranes with [32P]-NAD+ in the presence of cholera toxin is shown in Fig. 1 (young rats) and Fig. 2 (aged rats). The pattern of ribosylation was different between the two groups. Two cholera toxin-specific bands (M, 42,000 and 44,000) were ribosylated in the preparations from young rats (Fig. 1) whereas in old rats, only 1 band (M, 42,000) was clearly observed. The incorporation of 32P into the M, 42,000 band started at 30 min, and the ribosylation pattern of this band remained essentially unchanged in membranes from aged rats. In membranes from young rats, however, some ‘*P incorporation into the M, 42,000 and 44,000 bands was observed as early as 7.5 min. The ribosylation increased with time and by 75-90min these two bands had become highly ribosylated (Fig. 1 and Table 2). Also, at each time point, the extent of ribosylation was lower in the aged membranes than in those from the young rats (Table 2). DISCUPSION
We show that adenylate cyclase activity was significantly lower (p < 0.01) in the submandibular salivary glands of aged rats than in those of young rats. This reduction in aged rats was reflected in the basal and in the stimulated enzyme activity. As Mn*+ is known to increase the intrinsic activity of the catalytic unit of the enzyme (Florio and Ross, 1983) we measured the enzyme activities in the presence of this metal ion. The addition of Mn*+ resulted in about a 5-fold increase in the basal activity in membranes from young rats as compared to a 3.2-fold increase in membranes from aged rats. Fluoride-stimulated activity in membranes of aged rats was about 30% of that in membranes from young rats. As fluoride is known to stimulate adenylate cyclase activity by activating the G-proteins (Downs et al., 1980;
Howlett and Gilman, 1980; Kaslow et al., 1980), these findings suggest changes in the levels of Gproteins and/or in their interaction with the catalytic unit of the enzyme. In an attempt to obtain a better understanding of the mechanism(s) of age-related reduction in adenylate cyclase activity, we studied whether G-proteins were altered in ageing. The ribosylation of cholera toxin-specific G-proteins was lower in the submandibular glands of aged rats than in those of young rats. Whereas two distinct cholera toxinspecific bands (M, 42,000 and 44,000) were observed in the preparations from young rats, only one such band, M, 42,000, was clear in aged rats; the M, 44,000 band was very faint. The finding of lower fluoridestimulated enzyme activity in the membranes of aged rats is consistent with our finding of lower ribosylation of G,, suggesting reduced levels or activity of G, in these membranes from aged rats. Adenylate cyclase activity measured in the presence of forskolin and Mn*+ is considered to be a reasonably good index of the catalytic subunit (Seamon and Daly, 1981b; Daly, 1984; Ho and Shi, 1984). This activity in membranes of aged rats was 26% of that of the young rats. Therefore, it appears that the catalytic subunit of the enzyme may also be altered in the submandibular gland of aged rats. A decrease in forskolin-stimulated adenylate cyciase activity as a Table 2. Time-course for the incorporation of 32P into the cholera toxin-specific G-proteins in membranes Time of ribosylation (min) 1.5 15 30 45 60 75 90
from submandibular young and aged rats Peak area counts
glands
of
x IO-’
Young
Aged
6.6 5.9 9.2 16.1 21.8 29.0 *
0.6 0.5 5.1 6.0 8.6 9.7 9.3
Lubrol extracts obtained from membranes of young (3-months-old) and aged (24. months-old) Fisher 344 rats were analysed by SDSPAGE and autoradiography (see legend Fig. 1). The extent of incorporation of jzP into the cholera toxin-specific protein peptides (M, 42,000 and 44,000) as shown in Figs 1 and 2 was measured by densitometer. *The spot was too dense to be accurately measured.
Ageing and salivary gland adenylate cyclase result of ageing has also been observed
in other tissues
such as guinea pig ureter (Wheeler et al., 1986), and rat lymphocytes (Abrass and Scarpace, 1982), heart @‘Conner et al., 1983), erythrocytes (Farfel et al., 1984) and lung (Scarpace and Abrass, 1983). These studies have suggested that ageing may affect the catalytic unit of the enzyme because forskolin activates adenylate cyclase by direct binding to that unit (Clark et al., 1982; Seamon et al., 1981; Seamon and Daly, 198la). There is some evidence that functional G, is required for the full expression of forskolinstimulated adenylate cyclase activity (Darfler et al., 1982). As we found. lower ribosylation of G, in the submandibular salivary-gland membranes of aged rather than young rats, the lower levels of forskolinstimulated and fluroride-stimulated adenylate cyclase activity may be at least partly related to the reduced levels of G-proteins in the membranes of aged rats. We did not examine the pertussis toxin-specific ribosylation of Gi because, in another study, we did not find any significant ribosylation of G-proteins with this toxin in rat submandibular gland (Ahmad, Alam and Alam, 1990). Further studies are needed to determine whether the lower adenylate cyclase activity and a reduction in the levels of G,, found in the membrane preparations from aged rats would result in functional changes in the gland such as in the secretion of salivary proteins. If so, this may compromise the protective action of saliva against oral disease. Acknowledgements-This
study was supported by National Institute of Health Grant No. DE 05978. We thank Linda Armstrong for manuscript preparation.
REFERENCES
Abrass I. B. and Scarpace P. J. (1982) Catalytic unit of adenylate cyclase: reduced activity in aged-human lymphocytes. J. Clin. Endocr. Metab. 55. 10261028. Ahmad- S. N., Alam S. Q. and Alam B.‘S. (1990) Influence of dietary omega-3 fatty acids on transmembrane signalling in rat submandibular salivary gland. Cell Signal. 2, 2941.
Alam S. Q. and Alam B. S. (1986) Effect of essential fatty acid deficiency on acyl group composition, membrane fluidity and adenylate cyclase activity in plasma membranes of rat submandibular salivary glands. J. Nutr. 116, 1620-1630. Baker S. P., March,md S., O’Neil E., Nelson C. A. and Posner P. (1985) Age-related changes in cardiac muscarinic receptors: Decreased ability of the receptor to form a high affinity agonist binding state. J. Geronr. 40, 141-146. Bodner L., Hoopes M. T., Gee M., Ito H., Roth G. S. and Baum B. J. (1983) Multiple transduction mechanisms are likely involved in calcium-mediated exocrine secretory events in rat parotid. cells. J. biol. Chem. 258, 2774-2777. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.
Clark R. B., Goka 1’. T., Green D. A., Barker R. and Butcher R. W. (1982) Differences in the forskolin activation of adenvlate ‘cyclase in wild-type and variant lymphoma cells.- Molec. Pharmac. 22, 869-613. Dalv J. W. (1984) Forskolin. adenvlate cvclase. and cell physiology: an overview. Ahv. Cyilic Nu>ieoiihe Protein Phosphorylation Rex. 17, 81-89.
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Dartler F. J., Mahan L. C., Koachman A. M. and Insel P. A. (1982) Stimulation by forskolin of intact S49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase. J. biol. Chem. 257, 11901-I 1907. Downs R. W. Jr, Spiegel A. M., Singer M., Reen S. and Aurbach G. D. (1980) Fluoride stimulation of adenvlate cyclase is dependent on the guanine-nucleotide regulatory protein. J. biol. Chem. 255. 949-954. Farfel 2. and Cohen Z. (1984) Adenylate cyclase in the maturing human reticulocyte: selective loss of the catalytic unit, but not of the receptor-cyclase coupling protein. Eur. J. Clin. Invest. 14, 7982. Feldman R. D., Limbird L. E., Nadeau J., Robertson D. and Wood A. J. J. (1984) Alterations in leukocyte /?adrenergic affinity with aging. A potential explanation for altered b-adrenergic sensitivity in the elderly. New Engl. J. Med. 310, 815-819.
Florio V. A. and Ross E. M. (1983) Regulations of the catalytic component of adenylate cyclase. Molec. Pharmat. 24, 195-202. Gee M. V., Ishikawa Y., Baum B. J. and Roth G. S.
(1986) Impaired adrenergic stimulation of rat parotid ccl1 glucose oxidation during aging. J. Geront. 41, 331-335. Ho R. J. and Shi Q. H. (1984) Evidence for a single forskolin-binding site in rat adipocyte membranes. Studies of [ 14,15-’Hldihydroforskolin binding and adenvlate cyclasd activation. j. biol. Chem. 259, 763&7636. _ Howlett A. G. and Gilman A. G. (1980) Hvdrodvnamic properties of the regulatory component bf adknylate cyclase. J. biol. Chem. 255, 2861-2866. Ito H., Baum B. J. and Roth G. S. (1981) /I-Adrenergic regulation of rat parotid gland exocrine protein secretion during aging. M&h. Age&g Dev. 15, 177-188. Ito H.. Baum B. J.. Uchida T.. Hoones M. T.. Bodner L. and’Roth G. S. ‘(1982) Modulation of rat parotid cell a-adrenergic responsiveness at a step subsequent to receptor activation. J. biol. Chem. 257, 9532-9538. Johnson G. L., Kaslow H. R. and Boume H. R. (1978) Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase. J. biol. Chem. 253, 712&7123. Kaslow H. R., Johnson G. L., Brothers V. M. and Boume H. R. (1980) A regulatory component of adenylate cyclase from human erythrocyte membranes. J. biol. Chem. 255, 373&3741. Kawai Y. and Arinze I. J. (1983) B-Adrenergic receptors in rabbit liver plasma membranes: predominance of &receptors and mediation of adrenergic regulation of hepaticglycogenolysis. J. biol. Chem. %I, 4364-4371. _ Kousvelari E. E.. Baneriee D. K.. Murtv L. and Baum B. J. (1988) N-linked-protein glycosylation in the rat parotid gland during aging. Mech. Ageing Dev. 42, 173-181.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature (Lond.) 227,
68&685.
Ludford J. M. and Talamo B. R. (1983) Independent regulation of g-adrenergic receptor and nucleotide binding proteins of adenylate cyclase. J. biol. Chem. 2S8, 483 l-4838. Narayanan N. and Tucker L. (1986) Autonomic interactions in the aging heart: age-associated decrease in muscarinic cholinergic receptor mediated inhibition of j-adrenergic activation of adenylate cyclase. Mech. Ageing Dev. 34, 249-259.
O’Conner S. W., Scarpace P. J. and Abrass I. B. (1983) Age associated decrease in the catalytic unit activity of rat myocardial adenylate cyclase. Mech. Ageing Dev. 21, 357-363.
Salomon Y., Londos C. and Rodbell M. (1974) A highly sensitive adenylate cyclase assay. Analyr. Biochem. 58, 541-548.
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Scarpace P. J. and Abrass I. B. (1983) Decreased fi-adrenergic agonist affinity and adenylate cyclase activity in the senescent rat lung. J. Geronr. 38, 143-147. Schmid G., Geiger H., Bahner U. and Heidland A. (1988) Glandular adenylate cyclase system in genetic hypertension: age-dependent response to catecholamines. Eur. J. Pharmac.
147, 397402.
Seamon K. B. and Daly J. W. (1981a) Activation of adenylate cyclase by the diterpene, forskolin does not require guanidine nucleotide regulatory proteins. J. biol. Chem. 256, 9799-9801.
et
a/.
Seamon K. B. and Daly J. W. (1981b) Forskolin: a unique diterpene activator of cyclic AMP-generating system. J. Cyclic Nucl. Res. 7, 201-224. Seamon K. B., Padgett W. and Daly J. W. (1981) Forskolin: Unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. natn. Acad. Sci. U.S.A. 78, 3363-3367. Wheeler M. A., Housman A., Cho Y. H. and Weiss R. M. (1986) Age dependence of adenylate cyclase in guinea pig ureter homogenate. J. Pharmac. exp. Ther. 239, 99-103.
1990 Copyright
0
0403-9969/90 $3.00 + 0.00 1990 Pergamon Press plc
EFFECT OF AGEING ON ADENYLATE CYCLASE ACTIVITY AND G-PROTEINS IN RAT SUBMANDIBULAR SALIVARY GLANDS* S. N. AHMAD,
S. Q. ALAM? and B. S. ALAM
Louisiana State University Medical Center, Department of Biochemistry and Molecular Biology, New Orleans, LA 70119, U.S.A. (Accepted 23 May 1990)
Summary-Membranes were prepared from the submandibular salivary glands of male young (3-monthsold) and aged (24-months-old), Fisher 344 rats, and assayed for adenylate cyclase activity and for the ribosylation of G-proteins in the presence of [32P]-NAD+ and cholera toxin. Adenylate cyclase activity (basal, fluoride-, forskolin-, and isoproterenol-stimulated) was significantly lower (p < 0.01) in the glands of aged rats. The pattern of ribosylation was different between the 2 groups. Two cholera toxin-specific bands (M, 42,000 and 44,000) were ribosylated in the glands of young rats whereas in aged rats, only an M, 42,000 band was clearly observed. The incorporation of 32P from NAD+ into the M, 42,000 and 44,000 bands increased with the time of ribosylation (7.5-90 min). In aged rats, however, the incorporation of 32P into the M, 42,000 band was much slower and did not increase much after 60 min of ribosylation. At each time point, the extent of ribosylation was lower in the membranes from the aged rats. These findings suggest that the decrease in adenylate cyclase activity in the submandibular salivary glands of aged rats may be due partly to the changes in their levels of G, as a result of ageing. Key words: salivary gland, adenylate cyclase, G-protein.
INTRODUCTION
The adenylate cyclase system consists of the receptor, G-proteins (stimulatory, G, and inhibitory, Gi) and the enzyme adenylate cyclase. There is evidence that some receptor systems which act through G-proteins are impaired in ageing @‘Conner, Scarpace and Abrass, 1983; Baker et al., 1985; Narayanan and Tucker, 1986; Feldman et al., 1984). Deficits in these systems may involve #changes in receptor number, the G-proteins, or the catalytic subunit of adenylate cyclase. An impaired stimulation of adenylate cyclase with age in rat heart was reported to result from a decrease in the amount of G, and not from a loss of B-adrenergic receptors @‘Conner et al., 1983). Similarly, in aged rat heart, the density of muscarinic cholinergic receptors was not changed, but these receptors formed fewer high-affinity binding complexes with Gi (Baker et al., 1985). Decreased coupling of the receptor to Gi has been shown to result in impaired muscarinic agonist inhibition of myocardial, p-adrenergic: receptor-stimulated, adenylate cyclase activity (Narayanan and Tucker, 1986). In leucocytes from aged humans, the density of /I-adrenergic receptors is not changed, but receptorstimulated adenylate cyclase activity is decreased because of impaired coupling between the receptor *Presented in part at the International Association for Dental Research/American
Association for
Dental
Research,
Cincinnati, Ohio, 7-l 1 March 1990. tTo whom correspondence should be addressed. Abbrezktions: G-protein, guanine nucleotide-binding regulatory protein; SDS-PAGE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis.
and G-proteins (Feldman et al., 1984). Such impaired coupling with age may result from an age-related loss of G-proteins which prevents formation of the high-affinity binding complex. In several systems, such as rat lymphocyte (Abrass and Scarpace, 1982), heart @‘Conner et al., 1983), erythrocytes (Farfel and Cohen, 1984), lung (Scarpace and Abrass, 1983), and guinea pig ureter (Wheeler et al., 1986), a significant decrease in the ability of forskolin to stimulate adenylate cyclase activity has been observed with ageing. As forskolin activates adenylate cyclase by direct binding to the catalytic unit of the enzyme (Clark et al., 1982; Seamon, Padgett and Daly, 1981; Seamon and Daly, 198la), these studies suggest age-related changes in that catalytic unit. Several aspects of stimulus-secretion coupling in rat parotid acinar cells during ageing have been characterized (Ito, Baum and Roth, 1981; Ito et al., 1982; Bodner et al., 1983; Gee et al., 1986). The /I-adrenoreceptor signal-transduction coupling is unchanged with age, with respect to exocrine protein secretion in rat parotid gland (Ito et al., 1981). However, Schmid et al. (1988) found that the response of isoproterenol, as measured by CAMP production and adenylate cyclase activity in parotid gland homogenates, was reduced in older (16-18 weeks) as compared to younger (6-8 weeks) rats. Kousvelari et al. (1988) have reported a decrease in the incorporation of [‘HI-mannose into N-linked glycoproteins in parotid acinar cells of aged rats. After ,!I-adrenergic activation, however, cells from both young adult and aged glands showed increased N-linked protein glycosylation to almost the same extent. 885
S. N.
886
hiMAD
To our knowledge there are no published reports of the effects of ageing on the fi-adrenergic-adenylate cyclase complex in the submandibular salivary gland. We have now investigated adenylate cyclase activity and the G-proteins in these glands of young and aged rats. MATERIALS AND METHODS
Male Fisher 344 rats were purchased from the National Institute on Aging (Bethesda, MD). Young rats were 3-months-old; the aged rats were 24months-old. According to the information provided by the National Institute, the rats had been tested for viral and mycoplasmal antibodies. No clinical or histopathological evidence of infectious illness was detected. The rats had been fed NIH 31, TEKLAD diet while housed at the Institute. Upon arrival in our laboratory, they were housed individually in wirebottomed cages and fed a standard laboratory diet (Purina rat chow) for about 1 week until they were killed. The body weight of the young rats was 190-275 g, and that of aged rats, 380490 g. After killing, the submandibmar salivary glands were dissected out, cleaned from the sublingual glands, and then crude membranes were prepared from them, as described previously (Ludford and Talamo, 1983; Ahmad, Alam and Alam, 1990). In brief, the glands were homogenized in 10 volumes (w/v) of ice-cold 10 mM tris-HCl, pH 7.4, containing 0.3 M sucrose and 0.5 mM EDTA in a Potter-Elvejhem glass homogenizer with 20-26 up and down strokes at 4°C and 3000 rpm; the resultant homogenate was then strained through two layers of cheese cloth and centrifuged for 10 min at 2000g. The pellet was washed twice by suspending it in 10 volumes of sucrose-tris medium and centrifuging (as above), and then by resuspension and centrifugation in 10 volumes of 50 mM tris and 0.05 mM EDTA, pH 7.4. The final pellet was suspended in sucrose-tris medium at a protein concentration of 5-10 mg/ml and stored in small portions in liquid nitrogen until analysis. For use, thawed membranes were washed once with 10 mM tris-HCI, pH 7.4, and the pellet resuspended in 5 volumes (per g wet wt) of 50mM potassium phosphate buffer, pH 7.3. Protein concentration in the membranes was measured in replicates of samples by Bradford’s method (1976) after solubilizing the membranes with NaOH as described by Kawai and Arinze (1983), using bovine serum albumin as standard. ADP-ribosylation of membrane proteins
ADP-ribosylation of membrane proteins was done with GI-[~~P]-NAD+ and cholera toxin, as described by Johnson et al. (1978) but with some modification.
et
al.
Cholera toxin was preactivated by incubation of 1 mg/ml protein for 10 min at 32°C in a medium containing 10mM tris, 50mM glycine, pH 8.0, 20 mM dithiothreitol, 50 mM NaCl, 0.2 mM EDTA and 0.6 mM sodium azide. Activated toxin was diluted into the final incubation mixture. The final labelling mixture (100 ~1) contained 50 mM potassium phosphate buffer, pH 7.3, 2OOpg membrane proteins, 10 mM thymidine, 20 mM arginine-free base, 5 mM ADP-ribose, 100 PM GTP, 5-10 FM cc-[32P]-NADC (sp. act. 25 Ci/mmol), 1Opg of toxin or toxin buffer. The reaction mixture was incubated for (r90min at 32°C. Blanks (membrane without cholera toxin) were incubated for 90min. Reaction was stopped at different times by adding 1 ml of ice-cold, 50 mM potassium phosphate buffer, pH 7.3, containing 10mM thymidine, 20mM arginine and 5 mM ADP-ribose. Tubes were kept on ice-cold water, vortexed and centrifuged at 20,OOOg for 30 min. All the supernatant was removed without disrupting the pellet. The pellet was resuspended in 60 ~1 of 1 mM HEPES, pH 8.0, containing 100pM MgCl,, 100pM EDTA and 0.7% lubrol PX. The membranes were dispersed with a Pasteur pipette and solubilized by allowing them to stand on ice with occasional vortexing. The samples were then centrifuged at 20,OOOg for 2 h and ADPribosylated proteins were examined in the supernatant. Eiectrophoresis
SDS-PAGE was carried out as described by Laemmli (1970) in 1.5 mm-thick slab gel using 10% acrylamide. Lubrol extract containing 60 pg protein was loaded on the gel and electrophoresed at a constant 70 V in the stacking and 200 V in the sieving gel. Gels were stained for 2 h in 0.1% Coomassie blue R-250, prepared in destaining solution (7% acetic acid and 10% methanol), destained, and dried under vacuum. Dried gels were exposed for 72 h to Kodak X-omat film for autoradiography. The intensity of 3zP incorporation was monitored on an image-array processor. Low molecular-weight standards were: bovine serum albumin, hen egg white ovalbumin, bovine carbonic anhydrase and soybean trypsin inhibitor. Adenylate cyclase assay
Adenylate cyclase activity was measured essentially according to the method of Salomon et al. (1974) by sequential chromatography on columns of Dowex cation-exchange resin and alumina to isolate [32P]-cAMP formed from a-[‘*PI-ATP by adenylate cyclase. Details of the procedure have been described by Alam and Alam (1986). In our study, 0.5 mM ATP was used as substrate. The recovery of reaction
Plate 1 Figs 1. and 2. Autoradiograms of lubrol extracts of [‘*PI-ADP-ribosylated membranes from submandibular salivary glands of young (Fig. 1) and aged (Fig. 2) rats. Membranes were incubated with [‘*PI-NAD+ in the absence (lane 1) or presence of cholera toxin (lanes 2-8) for different time periods and lubrol extracts were prepared. Proteins were separated on 10% acrylamide gel by SDS-PAGE and the gels were exposed to Kodak X-Omat film for 72 h. Molecular weight of marker proteins are shown to the left of the figure. Arrows indicate cholera toxin-specific changes in proteins M, 42,000 and 44,000 in young rats (Fig. 1) and M, 42,000 in aged rats (Fig. 2).
887
Ageing and salivary gland adenylate cyclase
7
a
t
+
+
45
60
75
90
1
2
-
t
-t
+
+
90
7.5
15
30
3
4
5
6
42.?-
Cholera Time
Toxin (min)
Fig. 1
1
2
3
4
5
6
7
8
-
+
+
t
t
t
t
•k
90
7.5
15
30
45
60
75
SO
42.7
31 .o
21.5
Cliolera Time
Toxin (min)
Fig. 2
Plate
I
888
S. N. AHMAD Table
I. Adenylate cyclase activity in submandibular (24-months-old)
Type rats
of
Young Aged
et al.
salivary glands of young (3-months-old) Fisher 344 rats
Basal -Mn’+
Basal +Mn*+
+ Fluoride*
10.5 * 0.3 4.6 k 0.5t
50.0 + 2.4 14.9 k 5.2f
558 f 26.8 174+ 11.ot
+ Forskolin* 350 * 19.4 89.5 & 3.5t
and aged
+ Isoproterenol’ 15Ok4.4 48.7 f 1.51
Values are mean + SEM of 3 separate assays, each done in triplicate. The enzyme activity is shown as pmol of cAMP/mg proteimmin. *The stimulated activity in the presence of fluoride (15 mM), forskolin (0.1 mM) or isoproterenol (20 PM) was measured in the presence of 10 mM MnCl,. Values with superscripts are significantly different from the corresponding values in young rats (Student’s t-test: tp < 0.001; QJ < 0.01).
product was monitored [3H]-cAMP.
by adding internal
standard,
RESULTS
Adenylate cyclase activity in the membranes from young and aged rats is shown in Table 1. The enzyme activity (basal, fluoride-, forskolin-, and isoproterenolstimulated) was significantly lower (p < 0.01 in most cases) in the membranes of aged rats. The non-stimulated (basal) activity in such membranes was 44% (- Mn*+ ) and 30% (+ Mn*+ ) of that in young rats. Similarly, the stimulated activities in the membranes of aged rats were 2633% of the corresponding activities in those of the young rats. The time-course for ribosylation of the membranes with [32P]-NAD+ in the presence of cholera toxin is shown in Fig. 1 (young rats) and Fig. 2 (aged rats). The pattern of ribosylation was different between the two groups. Two cholera toxin-specific bands (M, 42,000 and 44,000) were ribosylated in the preparations from young rats (Fig. 1) whereas in old rats, only 1 band (M, 42,000) was clearly observed. The incorporation of 32P into the M, 42,000 band started at 30 min, and the ribosylation pattern of this band remained essentially unchanged in membranes from aged rats. In membranes from young rats, however, some ‘*P incorporation into the M, 42,000 and 44,000 bands was observed as early as 7.5 min. The ribosylation increased with time and by 75-90min these two bands had become highly ribosylated (Fig. 1 and Table 2). Also, at each time point, the extent of ribosylation was lower in the aged membranes than in those from the young rats (Table 2). DISCUPSION
We show that adenylate cyclase activity was significantly lower (p < 0.01) in the submandibular salivary glands of aged rats than in those of young rats. This reduction in aged rats was reflected in the basal and in the stimulated enzyme activity. As Mn*+ is known to increase the intrinsic activity of the catalytic unit of the enzyme (Florio and Ross, 1983) we measured the enzyme activities in the presence of this metal ion. The addition of Mn*+ resulted in about a 5-fold increase in the basal activity in membranes from young rats as compared to a 3.2-fold increase in membranes from aged rats. Fluoride-stimulated activity in membranes of aged rats was about 30% of that in membranes from young rats. As fluoride is known to stimulate adenylate cyclase activity by activating the G-proteins (Downs et al., 1980;
Howlett and Gilman, 1980; Kaslow et al., 1980), these findings suggest changes in the levels of Gproteins and/or in their interaction with the catalytic unit of the enzyme. In an attempt to obtain a better understanding of the mechanism(s) of age-related reduction in adenylate cyclase activity, we studied whether G-proteins were altered in ageing. The ribosylation of cholera toxin-specific G-proteins was lower in the submandibular glands of aged rats than in those of young rats. Whereas two distinct cholera toxinspecific bands (M, 42,000 and 44,000) were observed in the preparations from young rats, only one such band, M, 42,000, was clear in aged rats; the M, 44,000 band was very faint. The finding of lower fluoridestimulated enzyme activity in the membranes of aged rats is consistent with our finding of lower ribosylation of G,, suggesting reduced levels or activity of G, in these membranes from aged rats. Adenylate cyclase activity measured in the presence of forskolin and Mn*+ is considered to be a reasonably good index of the catalytic subunit (Seamon and Daly, 1981b; Daly, 1984; Ho and Shi, 1984). This activity in membranes of aged rats was 26% of that of the young rats. Therefore, it appears that the catalytic subunit of the enzyme may also be altered in the submandibular gland of aged rats. A decrease in forskolin-stimulated adenylate cyciase activity as a Table 2. Time-course for the incorporation of 32P into the cholera toxin-specific G-proteins in membranes Time of ribosylation (min) 1.5 15 30 45 60 75 90
from submandibular young and aged rats Peak area counts
glands
of
x IO-’
Young
Aged
6.6 5.9 9.2 16.1 21.8 29.0 *
0.6 0.5 5.1 6.0 8.6 9.7 9.3
Lubrol extracts obtained from membranes of young (3-months-old) and aged (24. months-old) Fisher 344 rats were analysed by SDSPAGE and autoradiography (see legend Fig. 1). The extent of incorporation of jzP into the cholera toxin-specific protein peptides (M, 42,000 and 44,000) as shown in Figs 1 and 2 was measured by densitometer. *The spot was too dense to be accurately measured.
Ageing and salivary gland adenylate cyclase result of ageing has also been observed
in other tissues
such as guinea pig ureter (Wheeler et al., 1986), and rat lymphocytes (Abrass and Scarpace, 1982), heart @‘Conner et al., 1983), erythrocytes (Farfel et al., 1984) and lung (Scarpace and Abrass, 1983). These studies have suggested that ageing may affect the catalytic unit of the enzyme because forskolin activates adenylate cyclase by direct binding to that unit (Clark et al., 1982; Seamon et al., 1981; Seamon and Daly, 198la). There is some evidence that functional G, is required for the full expression of forskolinstimulated adenylate cyclase activity (Darfler et al., 1982). As we found. lower ribosylation of G, in the submandibular salivary-gland membranes of aged rather than young rats, the lower levels of forskolinstimulated and fluroride-stimulated adenylate cyclase activity may be at least partly related to the reduced levels of G-proteins in the membranes of aged rats. We did not examine the pertussis toxin-specific ribosylation of Gi because, in another study, we did not find any significant ribosylation of G-proteins with this toxin in rat submandibular gland (Ahmad, Alam and Alam, 1990). Further studies are needed to determine whether the lower adenylate cyclase activity and a reduction in the levels of G,, found in the membrane preparations from aged rats would result in functional changes in the gland such as in the secretion of salivary proteins. If so, this may compromise the protective action of saliva against oral disease. Acknowledgements-This
study was supported by National Institute of Health Grant No. DE 05978. We thank Linda Armstrong for manuscript preparation.
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