Effect of essential fatty acid deficiency on G-proteins, cAMP-dependent protein kinase activity and mucin secretion in the rat submandibular salivary glands

~

Cellular Signalling Vol. 7, No. 8, pp. 773-781, 1995. Copyright © 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0898-6568/95 $9.50 + 0.00

Pergamon 0898-6568(95)02005-M

EFFECT OF ESSENTIAL FATTY ACID DEFICIENCY ON G-PROTEINS, cAMP-DEPENDENT PROTEIN KINASE ACTIVITY AND MUCIN SECRETION IN THE RAT SUBMANDIBULAR SALIVARY GLANDS SYED Q. ALAM,* SELIM M. ABDEL-HAKIM, t"BASSIMA S. ALAM$ and I BRAHIM Y. I B R A H I M t Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1100 Florida Avenue, New Orleans, LA 70119 USA (Received 27 April 1995; and accepted 7 June 1995) A b s t r a c t - Studies were conducted to determine whether 13-adrenergic cell signalling is altered in submandibular

salivary glands (SMSG) is essential fatty acid (EFA) deficiency. Three groups of rats were fed diets which were deficient in EFA (EFAD), marginally deficient in EFA (MEFAD) or contained sufficient amount of EFA (Control). Rats were killed after 20 wk on diets, SMSG were dissected out and cyclic AMP-dependent protein kinase (PKA) activity was measured. The specific enzyme activities were higher in the homogenates and supernatant fractions of the gland from EFAD and MEFAD rats compared with the controls. The relative levels of guanine nucleotide-binding regulatory proteins (Gs and Gi) were also measured in the SMSG membranes of rats fed the 3 diets. The levels of Gs were significantly higher in the EFAD and MEFAD groups than in the controls. No significant differences were observed in the secretion of trichloroacetic acid-phosphotungstic acid (TCA-PTA) precipitable glycoproteins from the SMSG slices among the 3 dietary groups. Key words: EFA deficiency, Salivary glands, PKA activity, G-proteins, Mucin secretion.

INTRODUCTION

met [2]. In cystic fibrosis patients, there is a marginaldeficiency o f E F A [3,4]. There are also functional abnormalities in the exocrine glands [5-7]. Changes in physiological response to autonomic stimuli have been observed in salivary glands f r o m cystic fibrosis patients. McPherson et al. [8] reported decreased 13-adrenergic response in the submandibular salivary glands (SMSG), whereas Mangos [9] found increased 13-adrenergic and decreased cholinergic responsiveness in the parotid. In the labial glands no difference in agonist specificity or quantity o f K release was observed between the cystic fibrosis patients and the normal controls [I0]. The mechanism by which 13-adrenergic receptor stimulation leads to exocytosis by the intracellular increase in c A M P is not completely known. Cyclic A M P mediates most of its effects by activating cAMP-dependent protein kinase

Linoleic acid (18:2 n-6) and linolenic acid (18:3 n-3) are known to be essential fatty acids (EFA). E F A deficiencies have been observed in humans and can be induced in experimental animals. There is evidence suggesting that marginal E F A deficiencies m a y be prevalent during pregnancy and lactation [1], the two physiological conditions which increase the E F A requirements. This m a y be especially true in some of the developing countries where the fat intake is generally quite low (10-15°70 of the total calories) and the E F A requirements during pregnancy and lactation (5-7o70 of the total calories) are not adequately * Author to whom all correspondenceshould be addressed. tCurrent address: Department of Physiology, School of Medicine, E1-Minia University, Egypt. $ Deceased.

773

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s. Q. Alam et al.

(PKA). In the rat SMSG, 13-adrenergic receptor stimulation appears to be a requirement for mucin secretion [11-13]. Pure cholinergic or ~t-adrenergic receptor stimulation elicits only a slight response in mucin secretion from this gland [14]. The 13-adrenergic receptor-adenylate cyclase system consists o f the receptor, guanine-nucleotide binding regulatory proteins (Gs and G~) and the catalytic subunit of adenylate cyclase. The activation of this system results in a dramatic increase in intracellular c A M P levels [12, 15]. The c A M P pathway appears to be the predominant pathway which regulates secretion from the parotid and the submandibular gland. Previous studies from our laboratory have shown that the activities of some membraneassociated enzymes such as (Na + + K +) ATPase [16] and adenylate cyclase [17] are increased in the rat SMSG in EFA deficiency. An increase in adenylate cyclase activity which we observed in E F A deficiency may result in the modulation o f P K A activity and mucin secretion from the gland. Therefore, we have studied the effects of an E F A deficiency on P K A activity and on the secretion of high molecular weight glycoproteins from the rat SMSG. Guanine nucleotide-binding regulatory proteins (G-proteins) function as transducers in cellular signalling pathway. In a previous study [18] we found that their functional activity, as measured by ADP-ribosylation, was altered in the SMSG o f rats fed diets rich in n-3 fatty acids. Fluoride is known to increase adenylate cyclase activity by activating G-proteins [19, 20]. Since in our previous study [17] the fluoride-stimulated adenylate cyclase activity was higher in the SMSG of rats fed the EFA-deficient diets than in the controls, we have also investigated whether the levels o f G-proteins are altered or remain unaffected in E F A deficiency. METHODS Feeding study Male weanling Sprague-Dawley rats were fed standard laboratory ration for 3-4 days to acclimatise them to the laboratory conditions. Then they were weighed, randomly divided into 3 groups, and fed ad

libitum the following diets: Group I: EFA-deficient, 7070hydrogenated coconut oil (HCO); Group II: Marginally EFAD, 6.6070 HCO + 0.4°7o corn oil; Group III: Control, 7°7o corn oil. These diets provided 0, 0.5°7o and 9.6°/o of the total calories respectively from linoleic acid, as evaluated by the gas-liquid chromatographic analysis of the fatty acid composition of the oils (Table 1). Diets were based on AIN-76A [21,22]. Rats had free access to water. Rats were weighed once a week.

Preparation of gland homogenates, membranes and supernatants Rats were killed by decapitation after 20 wk on their respective diets. Heparinized blood samples were obtained and centrifuged at 2000 x g for 15 min to obtain plasma. The SMSG were dissected out, freed from the sublingual gland and homogenized in a medium containing 0.25 M sucrose, 50 mM tris buffer, pH 7.7 and 10 mM MgC12. The medium also contained protease inhibitors (leupeptin, 1 llM; pepstatin A, 0.05 pg/mL; phenylmethylsulfonyl fluoride, 100 ~tM; and trypsin inhibitor, 0.25 rtg/mL). The tissue was homogenized in a Potter Elvehjem homogenizer and filtered through 2 layers of cheese cloth. Aliquots of this homogenate were saved and stored under liquid nitrogen. However, most of the homogenate was used for the preparation of crude membranes and supernatant. For this purpose, the homogenate was centrifuged at 650 x g for 10 rain in a refrigerated centrifuge. The pellet was discarded, the supernatant was transferred to another tube and centrifuged at 37,000 x g for 30 min. The supernatant was aliquoted into 1 mL tubes and stored under liquid nitrogen. The pellet was resuspended in 10 mL of the homogenization medium and centrifuged again for 30 min at 37,000 x g. The pellet was suspended in the homogenization medium, aliquoted into 1 mL tubes and stored under liquid nitrogen. Aliquots of the homogenates, membranes and supernatant fractions were used for protein determinations using Bradford's method [23] using bovine serum albumin as standard. Fatty acid analyses Total lipids were extracted from aliquots of plasma and the SMSG homogenates by using the methodof Bligh and Dyer [24]. Internal standard (heptadecanoic acid) was added to an aliquot of the total lipid extracts and fatty acid methyl esters (FAME) were prepared by transesterification with boron trifluoride-methanol [25]. The FAME were purified by thin layer chromatography on 0.25 mm thick silica gel G plates and the fatty acid composition was determined

Signal transduction in EFA deficient submandibular salivary gland

775

Table 1. Fatty acid composition of the dietary fats

Fatty Acid Saturated 6:0 (caproic) 8:0 (caprylic) 10:0 (capric) 12:0 0auric) 14:0 (myristic) 16:0 (palmitic) 18:0 (stearic) Unsaturated 18:1 (oleic) 18:2 (linoleic) 18:3 (linolenic)

7% HCO (EFAD)

6.6°70HCO + 0.4% corn oil (MEFAD)

7070corn oil (Control)

0.5 7.1 6.2 50.9 17.8 8.0 7.4

0.5 6.7 5.9 48.0 16.8 8.2 7.1

11.6 1.7

0.8 0.2 -

2.1 3.6 0.1

23.1 60.0 1.0

Values are area percent, averages of 2 determinations. HCO = Hydrogenated coconut oil; - = not detected.

by gas chromatography as previously described [26]. Gas chromatography-mass spectrometry (GC/MS) was also used to confirm the identity of some FAME peaks. Details of the GC/MS method have been previously reported [27].

Adenylate cyclase assays Membranes (25 Ixg protein) from each group were assayed for adenylate cyclase activity by measuring the conversion of ct-[a2p]-ATP to [32p]-cAMP which was isolated by 2-step column chromatography as describedbySalomonetaL [28]. Detailsoftheadenylate cyclase assay have been previously described [ 17] except I mM ATP was used. The enzyme activity was measured without any exogenous stimulation (basal activity) and in the presence of isoproterenol (stimulated activity). The incubationmedium also contained 100 o.M dithiothreitol, 10 mM MnCI2 and 5 Ixg alamethicin (all final concentrations). Dithiothreitol is known to protect the sulfhydryl groups, Mn z+ increases the intrinsic activity of the catalytic unit of the enzyme [291 and alamethicin unmasks the latent adenylate cyclase activity [30].

cAMP-dependent protein kinase assays These assays were performed according to Roskoski's method [31]. Assays were performed in the presence and absence of 10 IxM cAMP. In brief, aliquots of SMSG homogenates, supernatant and membranes (50 Ixg protein) were incubated for 5 min' at 37°C in the presence of 0.1 mM kemptide, a serinecontaining peptide substrate (Sigma Chemical Co, St. Louis, MO), 50 mM MOPS buffer, pH 7.0, 10

mM MgCIE, 0.25 mg/mL bovine serum albumin, 0.75 mM isobutylmethylxanthine(IBMX), a phosphodiesterase inhibitor and 0.1 mM [~-32p]-ATP (all are final concentrations in the reaction mixture). The total volume of the reaction mixture was 50 IxL. Following incubation, 25 ~tL aliquots were withdrawn, spotted onto 1 × 2 cm phosphocellulose strips (Whatman p81) and immersed in 75 mM phosphoric acid to terminate the reaction. Blanks (the reaction mixture treated the same way except no homogenate) were added last. The strips were gently swirled in the acid for 2 min and the acid decanted. This washing procedure with phosphoric acid was repeated 3 more times. The phosphocellulose strips were dried in an oven at 100° C for approximately 5 min. The radioactivity was measured by liquid scintillation spectrometry after adding 10 mL of scintillationfluid. The specific activity of cAMP-dependent protein kinase was calculated as pmol [32p] incorporated/mg protein/min.

Immunoblotting G proteins (Gi and Gs) were quantitated by Western immunoblotting using commercially available rabbit antisera against synthetic peptides that correspond to defined regions of G proteins (one specific for the ¢t-subunit of the stimulatory G proteins, Gsct and another specific for ¢t-subunit of the inhibitory G proteins, Gict-1 and GiQ-2, Du Pont NEN, Boston, MA). Crude membrane proteins (20 txg) of the SMSG of rats fed the various diets were separated by discontinuous SDS-polyacrylamide gel electrophoresis (1207o acrylamide running gel, 407o acrylamide stacking gel)

776

S.Q. Alam et at.

as described by Laemmil [32]. The proteins were electrophoretically transferred onto membranes (ImmunLite, Bio-Rad, positively charged nitrocellulose). Blots were rinsed in Tris-buffered saline, TBS (20 mM Tris, pH 7.5, 500 mM NaC1), incubated with 5 °/0 milk powder (in TBS) for 45 min at room temperature to block the membranes. The membranes were washed with TTBS (TBS + 0.05070 Tween-20) for 10 rain and then incubated for 90 rain with an appropriate dilution of the antibody (1:175 for Gs and 1:600 for Gi) in antibody buffer. After washing with TTBS (5 times, for 5 min each), the membranes were incubated with 2nd antibody, goat anti-rabbit IgGalkaline phosphatase (1:3000 dilution in antibody buffer, Bio-Rad). The membranes were washed with TTBS (5 times, 5 min each), TBS (once for 5 min) and then incubated in Enhanced Chemiluminescent substrate solution for 5 min (Bio-Rad). The membranes were removed from the solution, covered with saran wrap and exposed to a film (Hyperfilm-MP, Amersham Corp.) for 3 to 20 min. The film was developed and the density of the bands for Gi and Gs was measured by a two-dimensional computerassisted laser densitometer.

SMSG Mucin Secretion Preparation of SMSG slices. Rats fed the EFAD, MEFAD and the control diet for 20 wk were killed, the SMSG were dissected out and rinsed with physiological saline. The sublingual glands were discarded and the SMSG were cut into thin slices with a razor blade in an incubation medium (IM) containing protease inhibitors (trypsin inhibitor, 0.01070 and aprotonin, 2 ~tg/mL). The IM contained (in mmol/L): NaC1, 116; KC1, 5.4; MgSO4, 0.81; Na2 HPO4, 1.01; CaC12, 1.8; glucose, 5.6; HEPES, 10 and 13-hydroxybutyrate, 1.0. The sliced tissue was washed twice with the IM. Preincubation. The sliced tissue (150 mg) was placed in a 20 mL conical flask containing 3 mL IM and was pre-incubated for 45 min at 37°C in a shaking water bath gassed with Oz. The sliced tissue was washed with the IM. [~4C]glucosamine incorporation into high molecular weight glycoproteins. The gland slices were incubated in 3 mL of the IM and 1 ~tCi/mL of [~4C] glucosamine for 1 h with shaking and gassing with 02 [11, 33]. The tissue was then washed 4 times with 3 mL IM containing glucosamine (1 mM). The tissue was incubated for 30 minutes in 3 mL of the IM containing glucosamine (lmM) and was washed 3 times with 3 mL IM. The tissue was incubated in 3 mL of the IM. Isoproterenol (10 ~tM) in ascorbic acid (0.1 mM) was added

to the IM and 200 txL aliquots were taken at 0, 30, 60, 90, and 120 min after adding the secretagogue. An aliquot was taken at 120 min for total protein determination [23] using a protein dye-binding reagent (Bio-Rad) and bovine serum albumin as a standard. The 200 ~tL aliquots were added directly to 2 mL ice-cold solution of 10070 trichloroacetic acid in 0.5070 phosphotungstic acid (10070 TCA-0.5070 PTA) containing glucosamine (4 mM). The formed precipitate was washed twice with 2 mL of 10070TCA-0.5 070PTA, hydrolyzed with 1 mL formic acid, 10 mL of universol was added and the radioactivity was measured in a 13-scintillation counter.

Statistical analysis The data were analysed using analysis of variance and the differences among the 3 dietary groups were calculated using Newman-Keul's test.

RESULTS B o d y weight gains o f rats fed the 3 diets for 20 w k are s h o w n in Fig. 1. These were significantly lower in r a t s fed the E F A D diet c o m p a r e d with the c o n t r o l s . T h e b o d y weight gains o f rats fed the m a r g i n a l l y E F A - d e f i c i e n t diet were i n t e r m e diate b e t w e e n t h o s e o f t h e c o n t r o l a n d the E F A D g r o u p s . Scaly d e r m a t i t i s , especially o n the tail, was also o b s e r v e d in rats fed the E F A D diet. T h e f a t t y acid c o m p o s i t i o n o f t o t a l lipids o f p l a s m a o f rats fed the 3 diets is shown in T a b l e 2. F a t t y a c i d p a t t e r n s c h a r a c t e r i s t i c o f a n E F A d e f i c i e n c y such as r e d u c t i o n in t h e levels o f 18:2 n-6 a n d 20:4 n-6 with c o n c o m i t a n t increase in the levels o f 18:1 a n d 20:3 n-9, were o b s e r v e d in the E F A deficient rat plasma. This resulted in higher r a t i o o f 20:3 n - 9 / 2 0 : 4 n-6 f a t t y acids in the E F A D g r o u p . A r a t i o o f greater t h a n 0.4 is c o n s i d e r e d to be a b i o c h e m i c a l i n d e x o f an E F A deficiency [34]. This r a t i o was 0.45 in the m a r ginally E F A - d e f i c i e n t rat plasma. Similar changes in the fatty acid concentrations in total lipid extracts o f the S M S G h o m o g e n a t e s were also observed in the E F A D and M E F A D rats (Table 3). T h e d a t a on a d e n y l a t e cyclase activities in the S M S G m e m b r a n e s ( T a b l e 4) s h o w e d no difference in b a s a l activity. H o w e v e r , in the presence

Signal transduction in EFA deficient submandibular salivary gland

777

700

600-

500 Z

I-:1:

400-

UJ >-

300"

o 200-

100"

~

~

~

~

1~

1~

1~

1'6

1~

2b

DURATION OF FEEDING (WEEKS) Fig. l . E f f e c t o f E F A deficiency o n b o d y w e i g h t g a i n s o f r a t s .

of isoproterenol, the enzyme activity was signifi-

homogenates

cantly higher in the EFAD

diets as compared

group compared

to

of rats fed the EFAD to the controls.

or MEFAD These diet-

related differences were more marked

the other 2 groups. T h e r e s u l t s o n P K A a c t i v i t i e s ( T a b l e 5) s h o w significantly h i g h e r e n z y m e activities in the S M S G

of PKA was the highest in the EFAD,

Table 2. Fatty acid composition of total lipids of plasma of rats fed Essential Fatty Acid Deficient (EFAD), Marginally Deficient (MEFAD) or Control Diets Fatty Acid 14:0 16:0 16:1 18:0 18:1 18:2 20:3 20:4 20:5 22:4 22:5 22:6 20:3 20:4

n-6 n-9 n-6

n-9/ n-6

EFAD 1.2 19.5 5.4 23.0 20.2 2.4 16.2 5.8 0.2 0.1 0.4 1.1

_ 0.1 a + 0.5" + 0.6 a + 1.3 a,t' + 1.1 a ± 0.1 a ± 0.5 a + 0.3 ~ _+ 0• ± 0a ± 0• +_ 0.1 ~

2.84 ± 0.23 a

in super-

natant fraction of the gland. The specific activity

MEFAD 1.2 19.5 3.4 25.4 14.7 7.2 5.9 13.7 0.2 0.2 0.4 1.0

+ 0.2 a ___ 1.0 a + 0.3 b + 1.0 b ± 1.0 b ± 0.3 b + 0.3 b ± 1.2 b -+ 0.0 ~ +_ 0.1 a ± 0.1 a -+ 0.1"

0.45 _+ 0.04 b

Control 0.2 ± 0 b 22.1 ± 1.0 a 1.6 +_ 1.6 c 19.8 + 1.0 • 10.8 ± 0.6 c 14.4 ± 0.3 c 0.3:1: lY 23.4 ± 1.2 c 0.2 _+ & 0.6 _ 0.1 b 0.5 ± 0.1 ~ 1.2 + 0.2 a 0.01 ± 0c

Values are area percent (mean + SEM o f 6 rats per group). Values with different superscripts in the same row are significantly different from each other (p < 0.05) using analysis of variance, Newman-Kenl's test.

followed

778

S . Q . Alam et al. Table 3. Fatty acid composition of total lipids of plasma of rats fed Essential Fatty Acid Deficient (EFAD), Marginally Deficient (MEFAD) or Control Diets Fatty Acid 14:0 16:0 16:1 18:0 18:1 18:2 n-6 20:2 n-9 20:4 n-6 20:5 22:4 22:5 22:6 20:3 n-9/ 20:4 n-6

EFAD 0.34 4.00 1.18 1.88 4.90 0.35 1.37 1.38 0.04 0.06 0.09 0.22

± 0.04 a _ 0.20 a ± 0.10 a ± 0.05 a + 0.20 a ± 0.02 a _+ 0.10 a ± 0.08 a ± 0~ + 0.02 a + 0a ± 0.01 a

1.00 _+ 0.02 a

MEFAD 0.41 ± 4.95 ± 1.00 ± 2.27 ± 4.60 ± 0.89 + 0.64 + 2.59 ± 0.06 + 0.10 ± 0.09 ± 0.19 +

0.05 a 0.40 b 0.10 b 0.10 b 0.50 a 0.04 b 0.02 b 0.10 b 0b 0.01 b 0.01 ~ 0.03 a

0.25 _+ 0.01 b

Control 0.14 4.77 0.52 2.22 2.77 2.70 0.11 3.27 0.05 0.20 0.15 0.13

+ 0.01b + 0.10 b + 0.05 b + 0.04 b + 0.20 b ± 0.10 c + 0c _+ 0.05 ~ + 0a + 0~ ± 0.01b ± 0.03 a

0.03 _+ 0c

Values are mg/g, mean ± SEM of 6 rats per group. Values with different superscripts in the same row are significantly different from each other (p < 0.05) using analysis of variance, Newman-Keul's test.

by the MEFAD and then the control group. S i m i l a r d i f f e r e n c e s w e r e o b s e r v e d in t h e a b s e n c e or presence of exogenous cAMP. The enzyme

M E F A D g r o u p s as c o m p a r e d t o t h e c o n t r o l s . T h e relative intensity f o r the Gi b a n d was also s o m e w h a t h i g h e r , b u t n o t s i g n i f i c a n t l y , in t h e

activity was t h e lowest in the c r u d e m e m b r a n e

2 e x p e r i m e n t a l g r o u p s as c o m p a r e d

fraction and there was no diet-related difference

control group. The secretion of ~4C-glucosamine labelled

among the 3 groups. D a t a o n G p r o t e i n s is s h o w n i n T a b l e 6. T w o b a n d s for Gs (44,000 M r a n d 48,000 Mr) a n d one b a n d f o r G i ( 4 1 , 0 0 0 M r ) w e r e o b s e r v e d in t h e

with the

h i g h m o l e c u l a r w e i g h t g l y c o p r o t e i n s i n t o t h e inc u b a t i o n m e d i u m was essentially similar in the

SMSG membranes prepared from each of the 3

S M S G slices p e r t a i n i n g t o t h e 3 g r o u p s ( T a b l e 7). N o s i g n i f i c a n t d i f f e r e n c e s w e r e o b s e r v e d i n

groups. The relative intensity of these bands was significantly higher for Gs (44,000Mr and 48,000

i s o p r o t e r e n o l - s t i m u l a t e d m u c i n s e c r e t i o n at a n y time point between the control group and the 2

Mr) in m e m b r a n e s p e r t a i n i n g to the E F A D a n d

experimental groups.

Table 4. Adenylate cyclase activities in SMSG membranes of rats fed EFAD, MEFAD and Control Diets Dietary Group EFAD MEFAD Control

Basal

+ 10 ~tM isoproterenol

32.3 + 5.4 a 22.5 + 1.4a 29.0 _+ 3.9 a

59.5 _+ 2.7 a 37.5 + 1.9b 46.7 +_ 3.8 b

Values are mean _+ SE of 6 assays in each group. The enzyme activity is expressed in p mol cAMP/mg protein/ rain. Values with different superscripts in a column are significantly different (p < 0.05) from each other using analysis of variance, Newman-Keul's test.

779

Signal transduction in E F A deficient s u b m a n d i b u l a r salivary gland Table 5. c-AMP dependent protein kinase activity in SMSG homogenates, supernatants and membranes of rats fed E F A D , M E F A D a nd control diets Homogenate

Supernatant

Membranes

Dietary Group

- cAMP

+ cAMP

- cAMP

+ c A MP

- c A MP

+ c A MP

EFAD MEFAD Control

176 ± 9 a 180 ± 7 a 134 ± 6 b

287 ± 14 a 313 ± 10 a 238 ± 7 b

474 ± 14 a 329 ± 32 b 221 ± 12 c

804 ± 38 a 589 ± 19b 423 ± 19~

82 ± 2 a 89 ± 2 ~ 90 ± 3 a

76 ± 3a 84 ± 2 a 84 ± 3~

Enzyme activity units are in p m o l / m g p r o t e i n / m i n . The values are me a n ± SE of quadruplicate assays in each group. The enzyme assays were repeated 3 times, with similar results. Values with different superscripts in a column are significantly different (p < 0.05) using analysis of variance, Newman-Keul's test. W h e n added, the c A M P concentration was 10 ~tM.

DISCUSSION The feeding o f EFAD and marginally EFAD diets resulted in growth retardation and changes in the fatty acid patterns o f plasma total lipids which are considered to be typical o f an EFA deficiency. An increase in the levels 5,8,11eiconsatrienoic acid (20:3 n-9), with a corresponding decrease in those o f arachidonic acid (20:4 n-6) was also observed in the SMSG total lipids of rats fed the E F A D and M E F A D diets. This resulted in 20:3 n-9/20:4 n-6 ratios of 1.0 in the EFAD and 0.25 in the M E F A D groups. A ratio o f 0.4 or higher is considered to be a biochemical index of an EFA deficiency [34]. Isoproterenol-stimulated adenylate cyclase activity was higher in the SMSG membranes o f rats fed the EFAD diet compared with the controls. This may be due to higher number o f 13-adrenergic receptor binding sites which we have previously observed in the rat SMSG in EFA deficiency [17]. It is not clear why there was Table 6. Levels of G-proteins in SMSG membranes of rats fed E F A D , M E F A D and control diets Gs Dietary Group

44,000 Mr

48,000 Mr

Gi

EFAD MEFAD Control

160 ± 16 132 ± 6 100

242 ± 47 212 ± 25 100

130 ± 13 145 ± 39 100

Values are mean _+ SE of 4 assays/group. The G-proteins were identified by Western i m m u n o b l o t t i n g using specific antibodies. Their levels were quantitated by densitometry. Values shown are relative to those of the control group.

no significant difference between the MEFAD and the Control groups in adenylate cyclase activity especially when the levels of Gs (44,000 Mr and 48,000Mr) and PKA activities were higher in both the E F A D and M E F A D groups as compared with the control group. We also measured the cAMP-phosphodiesterase activity in the SMSG homogenates and supernatant fractions of rats fed the 3 diets. No significant differences were observed among the 3 groups. The values for homogenates were (p m o l / m g protein/min) 52.7 in EFAD, 56.6 in MEFAD and 53.0 in the control group. The corresponding values in the supernatant were 66.5 in EFAD, 71.1 in MEFAD and 63.0 in the control group. The values are averages of 2 determinations per group, each performed in triplicate. There is very little information in the literature regarding the effects of malnutrition on 13-adrenergic receptor-stimulated signal transduct±on. A decrease in glucagon-stimulated cAMP signal transduction in isolated rat hepatocytes has been observed in protein-energy malnutrition [35]. This lack of responsiveness to glucagon was observed despite a greater hormone-stimulated cAMP production [36]. The stimulation o f adenylate cyclase with forskolin, guanine nucleotides or manganese in hepatic membranes was also enhanced. Also, the quantity of Gs was 70-80G70 greater in hepatocytes from malnourished rats, but the Gi levels were not altered. The activity of PKA was 60°10 lower in liver homogenates from malnourished rats compared with controls. A decrease in 13-adrenergic

780

S.Q. Alam et al. Table 7. Secretion of [~4C] glucosamine-labelled high molecular weight glycoproteins in SMSG slices of rats fed EFAD, MEFAD and Control diets

Dietary Group EFAD MEFAD CONTROL

Minues after adding isoproterenol, 10 ~tM 30 60 90 120 3.0 _+ 0.7 a 3.6 _+ 0.4 a 3.8 _+ 0.7 a

5.3 + 1.3a 6.6 _+ 0.8 a 6.3 _+ 1.4a

7.1 _+ 1.9a 9.3 _+ 1.14 8.6 + 2.0 a

9.2 _+ 2.4 a 11.3 _+ 1.5a 10.9 + 2.44

Data are expressed as number of fold increase (over time zero) in [14C] glucosamine-labelled high molecular weight glycoprotein secretion (mean _+ SEM of 5 rats/group). EFAD = essential fatty acid deficient. MEFAD = marginally deficient in essential fatty acids. Values with different superscripts are statistically different (p < 0.05) from each other using analysis of variance, Newman-Keurs tests.

r e c e p t o r d e n s i t y has been o b s e r v e d in the rat S M S G in m o d e r a t e p r o t e i n d e f i c i e n c y [37]. T h e P K A activity was m a r k e d l y h i g h e r in hom o g e n a t e s a n d s u p e r n a t a n t f r a c t i o n s o f the S M S G f r o m rats fed E F A D a n d M E F A D diets t h a n the c o n t r o l diet. T h e P K A a c t i v i t y was c o m p a r a t i v e l y l o w e r in the m e m b r a n e f r a c t i o n s a n d there was no d i e t - r e l a t e d difference. T w o different i s o z y m e s o f P K A ( t y p e I a n d t y p e II) have b e e n i d e n t i f i e d a n d c h a r a c t e r i z e d in salivary g l a n d s [38-40]. B o t h are a c t i v a t e d b y the b i n d i n g o f c A M P to the r e g u l a t o r y s u b u n i t which results in a d i s s o c i a t i o n o f the h o l o e n z y m e to a regulat o r y s u b u n i t - c A M P c o m p l e x a n d an active catalytic s u b u n i t . T h e free c a t a l y t i c s u b u n i t then p h o s p h o r y l a t e s a n u m b e r o f cellular p r o t e i n s . In the p r e s e n t study, we d i d n o t t r y to distinguish b e t w e e n these 2 isozymes. It w o u l d be interesting to s t u d y w h e t h e r E F A d e f i c i e n c y has a similar o r d i f f e r e n t effects o n t h e a c t i v a t i o n o f t y p e I a n d t y p e II f o r m s o f P K A . T h e r e is evidence t h a t in r a t liver P K A I a n d II a r e d i f f e r e n t l y r e g u l a t e d b y diet [41]. It was r a t h e r s u r p r i s i n g to o b s e r v e t h a t in spite o f h i g h e r P K A activity, the secretion o f m u c i n f r o m the S M S G slices was n o t altered b y E F A deficiency. Since t h e r e is a direct c o r r e l a t i o n b e t w e e n the a c t i v a t i o n o f P K A a n d m u c i n s e c r e t i o n in the rat S M S G [42, 43], one w o u l d have e x p e c t e d higher r a t e o f m u c i n secretion f r o m the S M S G o f E F A D a n d M E F A D rats. H o w e v e r , we h a v e o b s e r v e d similar p h e n o m e n a

in c A M P - m e d i a t e d system in rat h e a r t [44]. In E F A deficiency, the a d e n y l a t e cyclase activity a n d P K A activities were s i g n i f i c a n t l y reduced. H o w e v e r , t h e r e was no significant d i f f e r e n c e in 13-adrenergic responsiveness o f i s o l a t e d h e a r t p r e p a r a t i o n s f r o m rats fed the 3 diets. T h e results o f o u r p r e s e n t i n v e s t i g a t i o n t h a t the secretion o f high m o l e c u l a r weight g l y c o p r o teins f r o m t h e S M S G was n o t a l t e r e d in E F A deficiency a r e consistent with o u r p r e v i o u s s t u d y in which the a m y l a s e activity o r the t o t a l p r o t e i n c o n c e n t r a t i o n s in w h o l e - s t i m u l a t e d saliva were n o t altered in E F A d e f i c i e n c y [45]. T h e o n l y d i f f e r e n c e was a decrease in the f l o w rate o f saliva in E F A - d e f i c i e n t rats. Acknowledgements - This work was supported by NIH grant DE 05978 and the Egyptian Ministry of Higher Education. We thank Ms. Hau Tang for her technical assistance. REFERENCES 1. Holman R. T., Johnson S. B. and Ogburn P. L. (1991) Proc. Natl. A c a d . Sci. U S A 88, 48354839. 2. Robillard P. Y. and Christon R. (1993) Prostaglandins L e u k o t r i e n e s a n d Essential F a t t y A c i d s

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