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Adrenoceptor coupling mechanisms which regulate salivary secretion during aging
Life Sciences, Vol. 53, pp. 1873-1878 Printed in the USA
Pergamon Press
ADRENOCEPTOR COUPLING MECHANISMS WHICH REGULATE SALIVARY SECRETION DURING AGING
Atsushi Miyamoto*, Tsunehisa Araiso**, Tomiyasu Koyama** and Hideyo Ohshika Department of Pharmacology, School of Medicine, Sapporo Medical University, South 1, West 17, Chuo-ku, Sapporo 060, Japan and **Laboratory of Molecular Physiology, Research Institute of Applied Electricity, Hokkaido University, North 12, West 6, Kita-ku, Sapporo 060, Japan (Received in final form October 11, 1993)
Summary In parotid slices and membranes from Wistar rats 2, 12 and 24 months old, changes are noted in adrenoceptor-stimulated K ÷ fluxes, formation of [3H]inositol phosphates ([3H]IPs), cAMP production, and membrane environment. Norepinephrine-stimulated K ÷ efflux and formation of [3H]IPs in the slices proceed through an ch-adrenergic mechanism and are reduced 20% and 40% during aging, respectively. In 13-adrenoceptor stimulation with isoproterenol, no age changes were observed in K ÷ influx and cAMP production. The cholesterol content in membranes was reduced with age; concomitantly, the membrane viscosity decreased with age. These results indicate that the alterations in the membrane environment may provide agedependent modulation of cq-adrenoceptor coupling mechanisms and their functions. It is generally accepted that declines in several responses to hormones and drugs are a normal consequence of aging (1). This phenomenon is well documented for the responses mediated by activation of adrenoceptors. This loss of eq- and [3-adrenergic responsiveness occurs in a variety of cell types, including parotid cells (2,3), cardiac ventricle (4-6), aorta (7,8) and striata (9) from aged rats. Salivary secretion is mediated by the autonomic nervous system, which regulates the water, ionic and protein composition of the saliva. In particular, adrenergic signal transduction pathways are primarily responsible for the stimulation of protein synthesis and secretion in salivary cells. In in vitro experiments using rat parotid slices, the effects of adrenergic stimuli on K ÷ fluxes have been distinctly different for cq- and 15~-adrenoceptors, the former increasing K ÷ efflux, which is secondary to intracellular Caa÷ mobilization (10), and the latter decreasing the basal efflux of ions, which is mediated via intracellular cAMP production (11). Rat parotid glands are a useful tool to study exocrine hormone/receptor and signal transduction interactions. We have recently reported that the alterations in membrane environment as a function of * To whom correspondence should be addressed. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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age may modulate eq-adrenoceptor-G protein coupling/uncoupling in rat ventricular m y o cardium (5,6) and rat cerebral cortex (12). The current study focused on aging-dependent changes in the parotid adrenoceptor system and membrane environment. We report that the age-related changes in ctl-adrenergic secretory responsiveness are mediated, at least in part, by impaired IPs production and/or by alterations in membrane viscosity or composition. Materials and Methods
Animals: Male, Wistar strain rats (2, 12 and 24 months old) were used in this study. All animals were obtained from Japan Clea Co. and were maintained in a specific-pathogen-free room with water ad libitum until the experiments. Parotid glands were removed under pentobarbital (40 mg/kg, i.p.) anesthesia, the fat trimmed away and rapidly cut into slices of approximately 1 mm 3. Measurement of K* fluxes: The K ÷ fluxes from the slices were measured by the method previously reported (10,11). Briefly, the slices were incubated for 30 min at 37°C in Hanks' balanced salt solution (HBSS) gassed with a 95% 02/5% CO 2 mixture. Then they were incubated for 5 min with the maximal dose of drugs or vehicle. K ÷ concentrations in aliquots of the medium and the slices were determined by a flame photometer (Coming) using a lithium internal standard. The K ÷ fluxes were expressed as the percent of total K ÷ in the slices. Measurement of [3H]inositol phosphates ([3H]IPs) formation: The formation of [3H]IPs from [aH]inositol in the slices was measured by the method previously reported (5,12). Briefly, the slices were placed in HBSS containing 10 mM LiCl, 1.25 ~tCi myo-[aH]inositol (Amersham), and l-norepinephrine (NE) or vehicle. Tubes were gassed with 95% 02/5% CO2, capped, and then incubated at 37°C for 30 min. The reaction was terminated by cold HBSS containing 10 mM LiC1 and placing the tubes on ice. Each sample was homogenized and centrifuged at 10,000 xg for 10 min at 4°C. The supernatant fraction was recovered for measurement of [3H]IPs, and tissue pellets were analyzed for protein. [3H]IPs were separated from [3H]inositol by Dowex AGlx8 column chromatography. ACSII (Amersham) was added and samples were counted (Beckman). Measurement of cAMP production: The cAMP production in the slices was measured by the method previously reported (11). Briefly, cAMP production in the slices was measured after a 5 min stimulation in HBSS containing 1-isoproterenol (ISO) or vehicle. After the stimulation, samples were immediately frozen with liquid nitrogen, cAMP was assayed by radioimmunoassay with a cAMP assay kit (Yamasa). Measurement of viscosity and cholesterol and phospholipid contents: The viscosity and cholesterol and phospholipid contents in parotid membranes was measured by the method previously reported (5,12). Parotid membranes were also prepared by a method previously reported (13). Briefly, the viscosity of membranes was determined by measurement of fluorescence anisotropy using the fluorophore, 1,6-diphenyl-l,3,5-hexatriene (DPH). Cholesterol in the membranes was assayed by cholesterol assay kit (cholesterol E-test, Wako). Total lipids were extracted from the membranes (3 mg of protein/ml) in CHCI~/CH3OH (2:1). Aliquots of lipid extract dissolved in CHCI3 were analyzed for total lipid phosphorus. Protein content measurement and statistics: Protein determinations were performed by the method of Lowry et al. (14) using bovine serum albumin as the standard. Data are presented as mean-+S.E. Statistical analysis was performed using Student's t-test (two-tailed).
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Results
The first aim of this experiment was to determine the effects of NE and ISO on K ÷ fluxes from the parotid slices during aging. The effects of nearly maximal doses (10 ~tM) of NE and ISO on K ÷ fluxes from the parotid slices from rats aged 2, 12 and 24 months are shown in Table 1 Changes in responses of NE and ISO to K ÷ fluxes in parotid slices obtained from 2 - , 12- and 2 4 - m o n t h - o l d rats K ÷ fluxes (% of total)
Age (months)
Vehicle
NE
ISO
2
5.75-*0.63
11.50-.0.72
0.82-*0.70
12
5.16-.0.52
10.36--0.81
0.13--0.62
24
4.78+-0.74
9.48-*0.68 *
-0.22--0.41
K+ fluxes were determined 5 min after the addition of vehicle, NE (10 IxM) or ISO (10 o.M). Values are expressed as the mean±S.E, of 5 to 6 different experiments. *p<0.05 vs. 2 months of age.
A
B
-l 4oo
1300
~o.~" 100
n~
¢-..Q o. •
I" 200 < ----
~_=
100
O'
, 6
5
4
"0 6
-Log[Norepinephrine]M
f
5
4
-Log[Isoproterenol]M Fig. 1
NE dose-response stimulation of [3H]IPs formation (A) and ISO dose-response stimulation of cAMP production (B) in parotid slices obtained from rats aged 2 (open column), 12 (hatched column) and 24 (closed column) months. Data are expressed as a percentage and pmol/mg protein above the basal (vehicle) value in the absence of NE and ISO, respectively. Values are expressed as the mean±S.E. of 5 to 6 different experiments. The basal values (dpm/mg protein) of [3H]IPs from each age group were: 2 months, 738±62; 12 months, 835±51; and 24 months of age, 767±43. The basal values (pmol/mg protein) of cAMP from each age group were: 2 months, 4.92±0.42; 12 months, 6.08±0.56; and 24 months of age, 6.23±0.87. *p<0.05, **p<0.01 vs. 2 months of age.
--
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Table 1. Compared with 2 - m o n t h - o l d rats, NE-stimulated K ÷ efflux was significantly (p<0.05) reduced by 20% in the slices of 2 4 - m o n t h - o l d rats. NE-stimulated K ÷ efflux was unchanged between 2 and 12 months of age. No age differences were observed in K ÷ influx following ISO stimulation. Dose-response for NE-stimulated formation of [3H]IPs and for ISO-stimulated cAMP production in the slices from rats of various ages are shown in Fig. 1. Significant stimulation when compared to control values (vehicle) was seen in all rats examined (2, 12 and 24 months of age) at concentrations of 1 ~tM NE and 1 IxM ISO or greater. An age-dependent significant decrease in the ability of NE to stimulate the formation of [aH]IPs was observed (p<0.05 and p<0.01)(Fig. 1, A), whereas the ISO-stimulated cAMP production did not change with age (Fig. 1, B). In rats 24 months of age, the EDso for NE to stimulate the formation of [3H]IPs was significantly (p<0.05) higher than those at 2 months of age (6.8-+0.5 [xM for 2 months, 7.4-+0.7 IxM for 12 months and 8.6-+0.4 ~tM for 24 months of age). The EDs0 for ISO to stimulate cAMP production was essentially identical (2.4-+0.3 gM for 2 months, 2.2-+0.3 ~tM for 12 months and 2.3-+0.4 ItM for 24 months of age). Table 2 Changes in the molar ratio of cholesterol to phospholipids and the viscosity in parotid membranes obtained from 2-, 12- and 2 4 - m o n t h - o l d rats
Age (months) Cholesterol (~tg/mg protein) Phospholipids (~tg/mg protein) C/P molar ratio
2
12
83.21-+6.58
75.03-+3.02
187.52-+18.36
183.34-+17.59
0.89-+0.04
0.82-+0.04
24 68.64-+2.45 * 179.03-+10.26 0.77-+0.05 *
r s value
0.134-+0.003
0.122-+0.005 *
0.100-+0.003 **
Viscosity qooise)
0.635-+0.013
0.551-+0.020 *
0.410-+0.014 **
r s is the steady-state fluorescence anisotropy measured at 37°C. Values are expressed as the mean-+S.E, of 5 to 8 experiments. *p<0.05, **p<0.01 vs. 2 months of age. The effects of aging on the molar ratio of cholesterol to phospholipids and viscosity in rat parotid membranes are presented in Table 2. The cholesterol contents in membranes from 24month-old rats were significantly (p<0.05) lower than those from 2 - m o n t h - o l d rats. In contrast, membrane phospholipid contents remained essentially unchanged during aging. An overall significant (p<0.05) decrease in the molar ratio of cholesterol to phospholipids was observed at 24 months of age. A similar trend was observed at 12 months of age; however, this was not statistically significant. As judged by computer analysis using the fluorophore DPH, r 8 values at 12 and 24 months of age were significantly (p<0.05 and p<0.01, respectively) lower than at 2 months of age. Similar significant (p<0.05 and p<0.01, respectively) differences were observed at 12 and 24 months of age when results were expressed as parameters such as the viscosity of the membranes. Discussion
Activation of Otl-adrenoceptors in rat parotid cells causes univalent-ion fluxes such as that of K ÷, which are secondary to cellular Ca 2÷ mobilization (15,16). It was recently suggested by
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1877
Ito et al. (17) that epinephrine was less effective in stimulating phosphoinositide turnover and K ÷ efflux in parotid cells from aged Wistar rats as compared to their young controls. We also found that K ÷ efflux and IPs formation in parotid tissues, evoked by Ctl-adrenoceptor stimulation, declined with age. It appears, therefore, that ch-adrenergic K ÷ efflux responsiveness in parotid tissues is consistently diminished by impaired IPs production during aging. Recently, we reported that a close relationship exists between Otl-adrenoceptor-stimulated IP3 production and Ca 2÷ mobilization and that reduced stimulation of IPa production may largely explain Ca 2÷ mobilization during aging (2). Consequently, these current results suggest that in senescent parotid tissue, the ability of Ca 2÷ to elicit K ÷ efflux is diminished. Such an effect of age may reflect differential cq-adrenoceptor coupling mechanisms or IP3 production following cqadrenoceptor stimulation (2,3,18). We have previously reported that the adenylyl cyclase/cAMP system is one of the biochemical mechanisms which participates in the regulation of K ÷ influx (11). Changes in cAMP levels in parotid tissues have been implicated in the regulation of salivary secretion (19), and various studies have reported decreased (20) or unaltered (21) I$-adrenergic function in aged rats. The former findings suggested that despite the unchanged characteristics of the 13adrenoceptors, impaired signal transduction may alter receptor- mediated salivary secretion in the aging parotid gland. The latter findings suggested that the rat parotid 15-adrenergic regulation of protein secretion (amylase release) remains functionally intact during aging. Such apparent discrepancies may be due to sex, strain or preparation differences. In the current work using male Wistar rats, we have demonstrated that no age changes were observed in K ÷ influx and cAMP production following 15-adrenoceptor stimulation. These results suggest that the rat parotid lS-adrenergic system remains functionally intact during aging. Earlier investigations from our laboratory have suggested that the alterations in membrane viscosity or composition as a function of age may be one of the mechanisms responsible for the altered cq-adrenoceptor binding sites or transduction of messages in cardiac and nervous tissues (5,12). Membrane viscosity experiments using DPH fluorescence showed that parotid membranes from aged animals were more fluid (p=0.41) than membranes from young animals (p=0.64) and both were much more fluid than cerebral cortex (p=l.01). This finding is consistent with our earlier reports on the relationship between eq-adrenoceptor signal transduction and membrane viscosity (5,12). The apparent decrease in parotid membrane viscosity during aging may be the result of a change in the molar ratio of cholesterol to phospholipids, not its cause. It is possible that the lower cholesterol content may result from either decreased synthesis or increased degradation of cholesterol (22). Since changes in membrane fluidity have been proposed to modulate Ctl-adrenoceptor/G protein interactions during development (23), they appear to be related to the altered rates of protein-protein interactions such as ch-adrenoceptor/G protein coupling. However, the possibility exists that the membrane characteristics themselves do not exert an important effect on the [3adrenoceptor/adenylyl cyclase coupling system in parotid glands and the exact mechanisms of the above phenomena during aging still remain to be clarified. In conclusion, the impaired K ÷ efflux ability of ctl-adrenoceptor stimulation during aging can be attributed to a diminished efficacy in oh-adrenoceptor/G protein coupling and subsequent reduced IPs formation. Lower membrane viscosity in parotid membranes of aged animals may also have an important influence to diminish a~-adrenoceptor/G protein interaction.
Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of
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Education, Science and Culture, Japan. References
1. G.S. ROTH, Endocrine Function and Aging, H.J. Armbrecht, R.M. Coe, and N. Wongsurawat (Eds), 26-34, Springer-Verlag, New York (1989). 2. A. MIYAMOTO, R. VILLALOBOS-MOLINA, M.A. KOWATCH and G.S. ROTH, Am. J. Physiol. 262 (Cell Physiol. 31) Cl181-Cl188 (1992). 3. R. VILLALOBOS-MOLINA, A. MIYAMOTO, M.A. KOWATCH and G.S. ROTH, Eur. J. Pharmacol. 226 (Mol. Pharmacol.) 129-131 (1992). 4. J.S. PARTILLA, M.T. HOOPES, H. ITO, E.M. DAX and G.S. ROTH, Life Sci. 31 2507-2512 (1982). 5. A. MIYAMOTO and H. OHSHIKA, Gen. Pharmacol. 20 647-651 (1989). 6. A. MIYAMOTO, S. KAWANA, H. KIMURA AND H. OHSHIKA, Eur. J. Pharmacol. (Mol. Pharmacol.) (in press) 7. L. HYLAND, P. WARNOCK and J.R. DOCHERTY, Naunyn-Schmiedeberg's Arch. Pharrnacol. 335 50-53 (1987). 8. J.R. DOCHERTY, Eur. J. Pharmacol. 146 1-5 (1988). 9. R. VILLALOBOS-MOLINA, J.A. JOSEPH and G.S. ROTH, Brain Res. 590 303-304 (1992) 10. A. MIYAMOTO and H. OHSHIKA, Japan. J. Pharmacol. 34 356-358 (1984). 11. A. MIYAMOTO and H. OHSHIKA, Japan. J. Pharmacol. 38 305-311 (1985). 12. A. MIYAMOTO, T. ARAISO, T. KOYAMA and H. OHSHIKA, J. Neurochem. 55 70-75 (1990). 13. A. MIYAMOTO, H. KIMURA and H. OHSHIKA, Gen. Pharmacol. 22 61-65 (1991). 14. O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 15. J.W. PUTNEY, Jr., J. Physiol. 268 139-149 (1977). 16. H. ITO, M.T. HOOPES, B.J. BAUM and G.S. ROTH, Biochem. Pharmacol. 31 567-573 (1982). 17. H. ITO, B.J. BAUM, T. UCHIDA, M.T. HOOPES, L. BODNER and G.S. ROTH, J. Biol. Chem. 257 9532-9538 (1982). 18. A. MIYAMOTO, M.A. KOWATCH and G.S. ROTH, Exp. Gerontol. 28 349-359 (1993) 19. M. SCHRAMM and E. NAIM, J. Biol. Chem. 245 3225-3231 (1970). 20. G. RAJAKUMAR and P.J. SCARPACE, J. Gerontol. 46 B249-B251 (1991). 21. H. ITO, B.J. BAUM and G.S. ROTH, Mech. Aging Dev. 15 177-188 (1981). 22. P.Y. POPPLEWELL and S. AZHAR, Endocrinology 121 64-73 (1987). 23. G. JAGADEESH, W-N. TIAN, S. GUPTA and R.C. DETH, Eur. J. Pharmacol. 189 (Mol. Pharmacol.) 11-21 (1990)
Pergamon Press
ADRENOCEPTOR COUPLING MECHANISMS WHICH REGULATE SALIVARY SECRETION DURING AGING
Atsushi Miyamoto*, Tsunehisa Araiso**, Tomiyasu Koyama** and Hideyo Ohshika Department of Pharmacology, School of Medicine, Sapporo Medical University, South 1, West 17, Chuo-ku, Sapporo 060, Japan and **Laboratory of Molecular Physiology, Research Institute of Applied Electricity, Hokkaido University, North 12, West 6, Kita-ku, Sapporo 060, Japan (Received in final form October 11, 1993)
Summary In parotid slices and membranes from Wistar rats 2, 12 and 24 months old, changes are noted in adrenoceptor-stimulated K ÷ fluxes, formation of [3H]inositol phosphates ([3H]IPs), cAMP production, and membrane environment. Norepinephrine-stimulated K ÷ efflux and formation of [3H]IPs in the slices proceed through an ch-adrenergic mechanism and are reduced 20% and 40% during aging, respectively. In 13-adrenoceptor stimulation with isoproterenol, no age changes were observed in K ÷ influx and cAMP production. The cholesterol content in membranes was reduced with age; concomitantly, the membrane viscosity decreased with age. These results indicate that the alterations in the membrane environment may provide agedependent modulation of cq-adrenoceptor coupling mechanisms and their functions. It is generally accepted that declines in several responses to hormones and drugs are a normal consequence of aging (1). This phenomenon is well documented for the responses mediated by activation of adrenoceptors. This loss of eq- and [3-adrenergic responsiveness occurs in a variety of cell types, including parotid cells (2,3), cardiac ventricle (4-6), aorta (7,8) and striata (9) from aged rats. Salivary secretion is mediated by the autonomic nervous system, which regulates the water, ionic and protein composition of the saliva. In particular, adrenergic signal transduction pathways are primarily responsible for the stimulation of protein synthesis and secretion in salivary cells. In in vitro experiments using rat parotid slices, the effects of adrenergic stimuli on K ÷ fluxes have been distinctly different for cq- and 15~-adrenoceptors, the former increasing K ÷ efflux, which is secondary to intracellular Caa÷ mobilization (10), and the latter decreasing the basal efflux of ions, which is mediated via intracellular cAMP production (11). Rat parotid glands are a useful tool to study exocrine hormone/receptor and signal transduction interactions. We have recently reported that the alterations in membrane environment as a function of * To whom correspondence should be addressed. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
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Adrenoceptor Coupling Mechanisms and Aging
Vol. 53, No. 25, 1993
age may modulate eq-adrenoceptor-G protein coupling/uncoupling in rat ventricular m y o cardium (5,6) and rat cerebral cortex (12). The current study focused on aging-dependent changes in the parotid adrenoceptor system and membrane environment. We report that the age-related changes in ctl-adrenergic secretory responsiveness are mediated, at least in part, by impaired IPs production and/or by alterations in membrane viscosity or composition. Materials and Methods
Animals: Male, Wistar strain rats (2, 12 and 24 months old) were used in this study. All animals were obtained from Japan Clea Co. and were maintained in a specific-pathogen-free room with water ad libitum until the experiments. Parotid glands were removed under pentobarbital (40 mg/kg, i.p.) anesthesia, the fat trimmed away and rapidly cut into slices of approximately 1 mm 3. Measurement of K* fluxes: The K ÷ fluxes from the slices were measured by the method previously reported (10,11). Briefly, the slices were incubated for 30 min at 37°C in Hanks' balanced salt solution (HBSS) gassed with a 95% 02/5% CO 2 mixture. Then they were incubated for 5 min with the maximal dose of drugs or vehicle. K ÷ concentrations in aliquots of the medium and the slices were determined by a flame photometer (Coming) using a lithium internal standard. The K ÷ fluxes were expressed as the percent of total K ÷ in the slices. Measurement of [3H]inositol phosphates ([3H]IPs) formation: The formation of [3H]IPs from [aH]inositol in the slices was measured by the method previously reported (5,12). Briefly, the slices were placed in HBSS containing 10 mM LiCl, 1.25 ~tCi myo-[aH]inositol (Amersham), and l-norepinephrine (NE) or vehicle. Tubes were gassed with 95% 02/5% CO2, capped, and then incubated at 37°C for 30 min. The reaction was terminated by cold HBSS containing 10 mM LiC1 and placing the tubes on ice. Each sample was homogenized and centrifuged at 10,000 xg for 10 min at 4°C. The supernatant fraction was recovered for measurement of [3H]IPs, and tissue pellets were analyzed for protein. [3H]IPs were separated from [3H]inositol by Dowex AGlx8 column chromatography. ACSII (Amersham) was added and samples were counted (Beckman). Measurement of cAMP production: The cAMP production in the slices was measured by the method previously reported (11). Briefly, cAMP production in the slices was measured after a 5 min stimulation in HBSS containing 1-isoproterenol (ISO) or vehicle. After the stimulation, samples were immediately frozen with liquid nitrogen, cAMP was assayed by radioimmunoassay with a cAMP assay kit (Yamasa). Measurement of viscosity and cholesterol and phospholipid contents: The viscosity and cholesterol and phospholipid contents in parotid membranes was measured by the method previously reported (5,12). Parotid membranes were also prepared by a method previously reported (13). Briefly, the viscosity of membranes was determined by measurement of fluorescence anisotropy using the fluorophore, 1,6-diphenyl-l,3,5-hexatriene (DPH). Cholesterol in the membranes was assayed by cholesterol assay kit (cholesterol E-test, Wako). Total lipids were extracted from the membranes (3 mg of protein/ml) in CHCI~/CH3OH (2:1). Aliquots of lipid extract dissolved in CHCI3 were analyzed for total lipid phosphorus. Protein content measurement and statistics: Protein determinations were performed by the method of Lowry et al. (14) using bovine serum albumin as the standard. Data are presented as mean-+S.E. Statistical analysis was performed using Student's t-test (two-tailed).
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Results
The first aim of this experiment was to determine the effects of NE and ISO on K ÷ fluxes from the parotid slices during aging. The effects of nearly maximal doses (10 ~tM) of NE and ISO on K ÷ fluxes from the parotid slices from rats aged 2, 12 and 24 months are shown in Table 1 Changes in responses of NE and ISO to K ÷ fluxes in parotid slices obtained from 2 - , 12- and 2 4 - m o n t h - o l d rats K ÷ fluxes (% of total)
Age (months)
Vehicle
NE
ISO
2
5.75-*0.63
11.50-.0.72
0.82-*0.70
12
5.16-.0.52
10.36--0.81
0.13--0.62
24
4.78+-0.74
9.48-*0.68 *
-0.22--0.41
K+ fluxes were determined 5 min after the addition of vehicle, NE (10 IxM) or ISO (10 o.M). Values are expressed as the mean±S.E, of 5 to 6 different experiments. *p<0.05 vs. 2 months of age.
A
B
-l 4oo
1300
~o.~" 100
n~
¢-..Q o. •
I" 200 < ----
~_=
100
O'
, 6
5
4
"0 6
-Log[Norepinephrine]M
f
5
4
-Log[Isoproterenol]M Fig. 1
NE dose-response stimulation of [3H]IPs formation (A) and ISO dose-response stimulation of cAMP production (B) in parotid slices obtained from rats aged 2 (open column), 12 (hatched column) and 24 (closed column) months. Data are expressed as a percentage and pmol/mg protein above the basal (vehicle) value in the absence of NE and ISO, respectively. Values are expressed as the mean±S.E. of 5 to 6 different experiments. The basal values (dpm/mg protein) of [3H]IPs from each age group were: 2 months, 738±62; 12 months, 835±51; and 24 months of age, 767±43. The basal values (pmol/mg protein) of cAMP from each age group were: 2 months, 4.92±0.42; 12 months, 6.08±0.56; and 24 months of age, 6.23±0.87. *p<0.05, **p<0.01 vs. 2 months of age.
--
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Table 1. Compared with 2 - m o n t h - o l d rats, NE-stimulated K ÷ efflux was significantly (p<0.05) reduced by 20% in the slices of 2 4 - m o n t h - o l d rats. NE-stimulated K ÷ efflux was unchanged between 2 and 12 months of age. No age differences were observed in K ÷ influx following ISO stimulation. Dose-response for NE-stimulated formation of [3H]IPs and for ISO-stimulated cAMP production in the slices from rats of various ages are shown in Fig. 1. Significant stimulation when compared to control values (vehicle) was seen in all rats examined (2, 12 and 24 months of age) at concentrations of 1 ~tM NE and 1 IxM ISO or greater. An age-dependent significant decrease in the ability of NE to stimulate the formation of [aH]IPs was observed (p<0.05 and p<0.01)(Fig. 1, A), whereas the ISO-stimulated cAMP production did not change with age (Fig. 1, B). In rats 24 months of age, the EDso for NE to stimulate the formation of [3H]IPs was significantly (p<0.05) higher than those at 2 months of age (6.8-+0.5 [xM for 2 months, 7.4-+0.7 IxM for 12 months and 8.6-+0.4 ~tM for 24 months of age). The EDs0 for ISO to stimulate cAMP production was essentially identical (2.4-+0.3 gM for 2 months, 2.2-+0.3 ~tM for 12 months and 2.3-+0.4 ItM for 24 months of age). Table 2 Changes in the molar ratio of cholesterol to phospholipids and the viscosity in parotid membranes obtained from 2-, 12- and 2 4 - m o n t h - o l d rats
Age (months) Cholesterol (~tg/mg protein) Phospholipids (~tg/mg protein) C/P molar ratio
2
12
83.21-+6.58
75.03-+3.02
187.52-+18.36
183.34-+17.59
0.89-+0.04
0.82-+0.04
24 68.64-+2.45 * 179.03-+10.26 0.77-+0.05 *
r s value
0.134-+0.003
0.122-+0.005 *
0.100-+0.003 **
Viscosity qooise)
0.635-+0.013
0.551-+0.020 *
0.410-+0.014 **
r s is the steady-state fluorescence anisotropy measured at 37°C. Values are expressed as the mean-+S.E, of 5 to 8 experiments. *p<0.05, **p<0.01 vs. 2 months of age. The effects of aging on the molar ratio of cholesterol to phospholipids and viscosity in rat parotid membranes are presented in Table 2. The cholesterol contents in membranes from 24month-old rats were significantly (p<0.05) lower than those from 2 - m o n t h - o l d rats. In contrast, membrane phospholipid contents remained essentially unchanged during aging. An overall significant (p<0.05) decrease in the molar ratio of cholesterol to phospholipids was observed at 24 months of age. A similar trend was observed at 12 months of age; however, this was not statistically significant. As judged by computer analysis using the fluorophore DPH, r 8 values at 12 and 24 months of age were significantly (p<0.05 and p<0.01, respectively) lower than at 2 months of age. Similar significant (p<0.05 and p<0.01, respectively) differences were observed at 12 and 24 months of age when results were expressed as parameters such as the viscosity of the membranes. Discussion
Activation of Otl-adrenoceptors in rat parotid cells causes univalent-ion fluxes such as that of K ÷, which are secondary to cellular Ca 2÷ mobilization (15,16). It was recently suggested by
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Ito et al. (17) that epinephrine was less effective in stimulating phosphoinositide turnover and K ÷ efflux in parotid cells from aged Wistar rats as compared to their young controls. We also found that K ÷ efflux and IPs formation in parotid tissues, evoked by Ctl-adrenoceptor stimulation, declined with age. It appears, therefore, that ch-adrenergic K ÷ efflux responsiveness in parotid tissues is consistently diminished by impaired IPs production during aging. Recently, we reported that a close relationship exists between Otl-adrenoceptor-stimulated IP3 production and Ca 2÷ mobilization and that reduced stimulation of IPa production may largely explain Ca 2÷ mobilization during aging (2). Consequently, these current results suggest that in senescent parotid tissue, the ability of Ca 2÷ to elicit K ÷ efflux is diminished. Such an effect of age may reflect differential cq-adrenoceptor coupling mechanisms or IP3 production following cqadrenoceptor stimulation (2,3,18). We have previously reported that the adenylyl cyclase/cAMP system is one of the biochemical mechanisms which participates in the regulation of K ÷ influx (11). Changes in cAMP levels in parotid tissues have been implicated in the regulation of salivary secretion (19), and various studies have reported decreased (20) or unaltered (21) I$-adrenergic function in aged rats. The former findings suggested that despite the unchanged characteristics of the 13adrenoceptors, impaired signal transduction may alter receptor- mediated salivary secretion in the aging parotid gland. The latter findings suggested that the rat parotid 15-adrenergic regulation of protein secretion (amylase release) remains functionally intact during aging. Such apparent discrepancies may be due to sex, strain or preparation differences. In the current work using male Wistar rats, we have demonstrated that no age changes were observed in K ÷ influx and cAMP production following 15-adrenoceptor stimulation. These results suggest that the rat parotid lS-adrenergic system remains functionally intact during aging. Earlier investigations from our laboratory have suggested that the alterations in membrane viscosity or composition as a function of age may be one of the mechanisms responsible for the altered cq-adrenoceptor binding sites or transduction of messages in cardiac and nervous tissues (5,12). Membrane viscosity experiments using DPH fluorescence showed that parotid membranes from aged animals were more fluid (p=0.41) than membranes from young animals (p=0.64) and both were much more fluid than cerebral cortex (p=l.01). This finding is consistent with our earlier reports on the relationship between eq-adrenoceptor signal transduction and membrane viscosity (5,12). The apparent decrease in parotid membrane viscosity during aging may be the result of a change in the molar ratio of cholesterol to phospholipids, not its cause. It is possible that the lower cholesterol content may result from either decreased synthesis or increased degradation of cholesterol (22). Since changes in membrane fluidity have been proposed to modulate Ctl-adrenoceptor/G protein interactions during development (23), they appear to be related to the altered rates of protein-protein interactions such as ch-adrenoceptor/G protein coupling. However, the possibility exists that the membrane characteristics themselves do not exert an important effect on the [3adrenoceptor/adenylyl cyclase coupling system in parotid glands and the exact mechanisms of the above phenomena during aging still remain to be clarified. In conclusion, the impaired K ÷ efflux ability of ctl-adrenoceptor stimulation during aging can be attributed to a diminished efficacy in oh-adrenoceptor/G protein coupling and subsequent reduced IPs formation. Lower membrane viscosity in parotid membranes of aged animals may also have an important influence to diminish a~-adrenoceptor/G protein interaction.
Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of
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Education, Science and Culture, Japan. References
1. G.S. ROTH, Endocrine Function and Aging, H.J. Armbrecht, R.M. Coe, and N. Wongsurawat (Eds), 26-34, Springer-Verlag, New York (1989). 2. A. MIYAMOTO, R. VILLALOBOS-MOLINA, M.A. KOWATCH and G.S. ROTH, Am. J. Physiol. 262 (Cell Physiol. 31) Cl181-Cl188 (1992). 3. R. VILLALOBOS-MOLINA, A. MIYAMOTO, M.A. KOWATCH and G.S. ROTH, Eur. J. Pharmacol. 226 (Mol. Pharmacol.) 129-131 (1992). 4. J.S. PARTILLA, M.T. HOOPES, H. ITO, E.M. DAX and G.S. ROTH, Life Sci. 31 2507-2512 (1982). 5. A. MIYAMOTO and H. OHSHIKA, Gen. Pharmacol. 20 647-651 (1989). 6. A. MIYAMOTO, S. KAWANA, H. KIMURA AND H. OHSHIKA, Eur. J. Pharmacol. (Mol. Pharmacol.) (in press) 7. L. HYLAND, P. WARNOCK and J.R. DOCHERTY, Naunyn-Schmiedeberg's Arch. Pharrnacol. 335 50-53 (1987). 8. J.R. DOCHERTY, Eur. J. Pharmacol. 146 1-5 (1988). 9. R. VILLALOBOS-MOLINA, J.A. JOSEPH and G.S. ROTH, Brain Res. 590 303-304 (1992) 10. A. MIYAMOTO and H. OHSHIKA, Japan. J. Pharmacol. 34 356-358 (1984). 11. A. MIYAMOTO and H. OHSHIKA, Japan. J. Pharmacol. 38 305-311 (1985). 12. A. MIYAMOTO, T. ARAISO, T. KOYAMA and H. OHSHIKA, J. Neurochem. 55 70-75 (1990). 13. A. MIYAMOTO, H. KIMURA and H. OHSHIKA, Gen. Pharmacol. 22 61-65 (1991). 14. O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 15. J.W. PUTNEY, Jr., J. Physiol. 268 139-149 (1977). 16. H. ITO, M.T. HOOPES, B.J. BAUM and G.S. ROTH, Biochem. Pharmacol. 31 567-573 (1982). 17. H. ITO, B.J. BAUM, T. UCHIDA, M.T. HOOPES, L. BODNER and G.S. ROTH, J. Biol. Chem. 257 9532-9538 (1982). 18. A. MIYAMOTO, M.A. KOWATCH and G.S. ROTH, Exp. Gerontol. 28 349-359 (1993) 19. M. SCHRAMM and E. NAIM, J. Biol. Chem. 245 3225-3231 (1970). 20. G. RAJAKUMAR and P.J. SCARPACE, J. Gerontol. 46 B249-B251 (1991). 21. H. ITO, B.J. BAUM and G.S. ROTH, Mech. Aging Dev. 15 177-188 (1981). 22. P.Y. POPPLEWELL and S. AZHAR, Endocrinology 121 64-73 (1987). 23. G. JAGADEESH, W-N. TIAN, S. GUPTA and R.C. DETH, Eur. J. Pharmacol. 189 (Mol. Pharmacol.) 11-21 (1990)