Inositol phosphates and intracellular calcium after bradykinin stimulation in fibroblasts from young, normal aged and Alzheimer donors

Neurobiologyof Aging, Vol. 12, pp. 469-473. ~ Pergamon Press plc, 1991. Printed in the U.S.A.

0197-4580/91 $3.00 + .00

Inositol Phosphates and Intracellular Calcium After Bradykinin Stimulation in Fibroblasts From Young, Normal Aged and Alzheimer Donors H . - M . H U A N G , * L. T O R A L - B A R Z A , * H. T H A L E R , t B. T O F E L - G R E H L * A N D G. E. G I B S O N *t

*The Burke Medical Research Institute, Department of Neurology and Neuroscience Cornell University Medical College, 785 Mamaroneck Avenue, White Plains, NY 10605 ~'Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, N Y 10021 R e c e i v e d 24 O c t o b e r 1990; A c c e p t e d 3 April 1991 HUANG, H.-M., L. TORAL-BARZA, H. THALER, B. TOFEL-GREHL AND G. E. GIBSON. lnositol phosphates and intracellular calcium after bradykinin stimulation in fibroblasts from young, normal aged and Alzheimer donors. NEUROBIOL AG1NG 12(5) 469-473, 1991.--Several studies suggest that alterations in the receptor-mediated phosphoinositide cascade and cytosolic free calcium concentration ([Ca2+]i) are involved in the pathophysiology of aging and Alzheimer's disease. Therefore, the phosphoinositide cascade and [Ca2+]i were determined under resting conditions and after stimulation with bradykinin (100 nM) in cultured human skin fibroblasts from young (21---3 years), normal aged (59---6 years) and Alzheimer subjects (58---6 years). The inositol polyphosphates (IP 3, IP2 and IP) were monitored after prelabeling the cells with [3H]inositol in serum free medium. [Ca2+]~ was determined with the fluorescent probe, fura-2AM, under exactly analogous conditions. The bradykinininduced formation of IP3 and IP2 increased significantly in fibroblasts from normal aged and Alzheimer donors compared to young subjects, but did not differ from each other. Bradykinin-induced IP 3 formation was 63-117% above the young group at time points between 10-60 s in normal aged or Alzheimer donors. Bradykinin-induced IP2 formation was 49-59% above the young group at time points between 10-60 s in normal aged or Alzheimer subjects. Neither the basal [Ca2+]i, nor the bradykininstimulated [Ca2+]i , differed among fibroblasts from young, normal aged and Alzheimer donors. The precise molecular basis and pathophysiological significance of the enhanced bradykinin-induced phosphoinositide cascade in fibroblasts from aged donors remains to be determined. Inositol phosphates Tissue culture

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THE receptor-mediated phosphoinositide cascade controls many cellular events and is closely linked to calcium homeostasis. Hydrolysis of phosphatidylinositol 4,5-bisphosphates (PIP2) generates two second messengers, inositol trisphosphates (IP3) and diacylglycerol. IP 3 stimulates the release of internal calcium stores (2,3), while diacylglycerol activates protein kinase C (19). IP 3 can be phosphorylated to inositol 1,3,4,5-tetrakisphosphate (25). IP 3 alone or in combination with inositol 1,3,4,5-tetrakisphosphate may modulate calcium entry (13,18). Several studies suggest that the receptor-mediated phosphoinositide cascade is involved in the pathophysiology of aging and Alzheimer's disease. However, the results between different laboratories and various tissue preparations are controversial. Brain phosphoinositide concentrations decline with age (27) and diminish further with Alzheimer's disease, which suggests a reduced phosphoinositide cascade (28). However, in human platelets, phosphoinositide turnover increases with age (1). Protein kinase C activity declines in flbroblasts from Alzheimer donors. Protein phosphorylation by intact fibroblasts (12), as well as in autopsied brains from Alzheimer patients (5), is altered in a manner that is consistent with diminished protein kinase C ac-

Fibroblasts

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tivity. The stimulation of the 13-adrenergic receptor produces more cyclic AMP in fibroblasts from Alzheimer patients than from controls (16), which may reflect an interaction of the regulation of cyclic AMP and protein kinase C (15,30). These resuits suggest that interactions between these two receptor-mediated signal transduction mechanisms may be altered in aging and Alzheimer's disease. Receptor-mediated phosphoinositide turnover has not been examined in tissues from Alzheimer patients. Both direct and indirect evidence suggests that calcium homeostasis is involved in the pathophysiology of aging and Alzheimer's disease (8). Calcium uptake by cultured skin fibroblasts diminishes with age, and is reduced further by Alzheimer's disease (20). Basal [Ca2÷]i and the response of [Ca2+]i to various growth factors have been reported to decrease with age in fibroblasts, and to decline further with Alzheimer's disease (21,22). Bradykinin, serum, 3,4-diaminopyridine and N-formyl-methionyl-leucyl-phenylalanine elevate [Ca 2÷ ]i transiently. The rate of the increase is slower, and the magnitude of the rise is less in fibroblasts from aged subjects, and is reduced further with Alzheimer's disease (22). However, other reports demonstrate that a serum-induced increase in [Ca 2+ ]i is exaggerated in fibro-

IRequests for reprints should be addressed to Dr. Gary E. Gibson.

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blasts from Alzheimer patients compared to age-matched controls (26). In addition, in human platelets, [Ca2+]~ increases with aging (10). The interactions between calcium and the phosphoinositide cascade may be altered in aging and Alzheimer's disease. The amount of calcium released from microsomes by IP 3 is reduced by age in rats' brains (4) and parotid cells (14). The interaction of receptor-mediated stimulation of phosphoinositide turnover and [Ca2+]i may mediate the changes due to aging and Alzheimer's disease. In the current studies, changes in the phosphoinositide cascade and in [Ca2+]i under resting conditions and after stimulation with bradykinin were determined in parallel in cultured human skin fibroblasts from young, normal aged and Alzheimer donors. METHOD

Chemicals and isotopes were from the indicated companies: Dulbecco's modified Eagle's (DME) media and fetal bovine serum (GIBCO; Grand Island, NY); D-myo[2-3H]inositol (20 Ci/ mmol, Amersham; Arlington, IL); bradykinin, ammonium formate, disodium tetraborate, sodium formate, HEPES, EGTA and LiC1 (Sigma Chemical, St. Louis, MO); anion-exchange resin AG1-X8 (formate, 200-400 mesh; Bio-Rad, Richmond, CA); liquid scintillation fluid Aquasol-2 (New England Nuclear, Boston, MA); fura-2 and fura-2AM (Molecular Probes; Eugene, OR); modular incubator chambers (Billups-Rothenberg, Delmar, CA). Human skin fibroblasts from apparently normal young persons (21_+3 years), normal aged subjects (59_+6 years) and from Alzheimer patients (58 _+6 years) were obtained from the Human Genetic Mutant Cell Repository at the Coriell Institute for Medical Research in Camden, NJ. No cell line from subjects listed as "at risk" for familial Alzheimer's disease was studied. All cell lines were studied at comparable cumulative population doubling time (17.7_+3.3) and at early passage numbers (10.6_+2.4). Thus age refers to chronological age of donor rather than age in culture. The cell lines that were used in these studies were from apparently normal young (GM3377, GM3651, GM2987, GM2937, GM0037 and GM4390), normal aged (AG4560, GM3524, AG6010, AG4440, AG9878 and AG8044) and Alzheimer donors (AG5809A, AG6265A, AG4402A, AG4400, AG6848 and AG7377). The fibroblasts were grown identically for the experiments on the phosphoinositide cascade and [Ca2÷] i. The conditions for prelabeling inositol phospholipids with [3H]inositol have been characterized (11). The cells were grown to confluence as a monolayer in DME media supplemented with 10% fetal bovine serum in T75 flasks. Seven days before the second messenger measurements, the cells were subcultured in 35 mm plastic petri dishes at a seeding density of 1 × l 0 4 cells/cm 2. Cells were grown for 3 days in 2 ml of DME media with 10% fetal bovine serum at 37°C in a humidified modular incubator that was aerated with 5% CO2 in 95% air. The media were then replaced with serum free DME media. One day later, the fluid was replaced with 2 ml of serum free DME media containing [3H]inositol (5 I*Ci/ml). After three days with the [3H]inositol, the media were replaced with fresh DME media containing 25 mM HEPES and 10 mM LiCI (pH 7.4) and the cells were incubated for one h at room temperature in a modular incubator. The cells were then incubated with fresh HEPES-LiC1-DME media at 37°C for 4 min. The media were changed and the cells were incubated with fresh 37°C HEPES-LiC1-DME media with or without bradykinin (100 nM) for the times indicated in Fig. 1. This concentration of bradykinin was chosen because our previous studies (11) indicate that this is an intermediate level that would

allow us to observe either an enhancement or a depression of the response. Incubations were terminated by aspirating the assay media and immediately adding 2 ml 0.2 N ice-cold percholoric acid (PCA). Procedures for extraction, neutralization and separation of inositol phosphates were essentially the same as previously described (11). In brief, the water-soluble products were eluted as follows: 15 ml of H20 to remove inositol, 15 ml of 5 mM disodium tetraborate in 60 mM sodium formate to elute glycerol phosphoinositol, 15 ml of 0.2 M ammonium formate in 0.1 M formate to elute IP, three 5 ml rinses of 0.4 M ammonium formate in 0.1 M formate to elute IP 2 and three 5 ml rinses of 1 M ammonium formate in 0.1 M formate to elute IP 3. A one ml aliquot was taken from each fraction and mixed with 10 ml of Aquasol-2 for measurement of radioactivity with a Beckman LS 5801 liquid scintillation counter. The lipids were extracted from the pellets with a modification (29) of the procedures of Eichberg and Hauser (6). Briefly, 2 ml of chloroform/methanol (2:1) and 0.5 ml of water were added and vigorously mixed for 20 s. After the lower organic phase was removed, the aqueous phase was extracted further with 1 ml of chloroform/methanol/12 N HCI (4:1:0.0125, by vol.). The neutral and acidic lipid extracts were combined and evaporated to dryness, and radioactivities were determined. Formation of each of the inositol phosphates was expressed as the fraction of [3H]inositol phosphates that were released from phospholipids [DPM in inositol phosphates/DPM in lipid extract]. This assumes that all of the inositol phosphates were produced from labelled phospholipids. The measurements of [CaZ+]i were the same as described previously (11). Fibroblasts were cultured exactly as for the experiments on inositol phosphates, except that the cells were seeded in petri dishes containing glass cover slips (25 mm), and the media did not contain [3H]inositol. On the day of the calcium measurements, the media were replaced with fresh serum free DME media containing 1% BSA, 25 mM HEPES (pH 7.4, room temperature) and 2 IxM fura-2AM. The cells were then incubated at room temperature for 1 h. The cells were rinsed three times with fresh incubation medium to remove any unincorporated fura-2AM. The coverslip was placed in the chamber of a temperature-controlled microincubator (Medical System Corp. Greenvale, NY), which was positioned on an inverted Olympus fluorescence microscope (model IMT2). Fresh DME media at 37°C were added, and the cells were allowed to equilibrate for 3 min. Resting values were monitored for 60 s, then the medium was replaced with the bradykinin-containing medium, and monitored for 3 min. Cytosolic free calcium was monitored by alternating the excitation wavelength between 355 and 378 nm 5 times a second with a delta scan (Photon Technology International; Princeton, NJ). The fluorescence of the cells was monitored with a photo-multiplier tube after passing through a 40 × Nikon UV objective and a 40 nm band pass filter with a peak at 509 nm. The ratio of the emitted fluorescence signal after excitation at 355 and 378 nm was used to calculate [Ca2+]i by the method of Grynkiewicz (9). Measurements were done on groups of cells (at least 5 cells) that were within a 4 × 4 ixm grid. Although this method permits a continuous monitoring of [Ca 2+ ]i, for comparison purposes values were calculated at 0 (i.e., basal), peak time, 30, 60 and 180 s after bradykinin stimulation. Statistics

Multivariate analysis of covariance (MANOVA) and analysis of variance (ANOVA) were used to analyze the results. Statistical significance (p<0.05) was determined by MANOVA on the ratio of DPM in each of the inositol phosphates to total phos-

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FIG. 1. Time course of bradykinin-induced accumulation of [3H]IP3 (A) and [aH]IP2 (B) in fibroblasts from young, normal aged and Alzheimer donors. Cells that had been prelabelled with [3H]inositol were incubated with or without bradykinin (100 nM) for 0, 10, 30 or 60 s. Values are mean +-SEM of two separate experiments in quadruplicate for six young, normal aged and Alzheimer cell lines. Significant differences (p<0.05) between groups were determined by ANOVA followed by Student-Newman-Keul's test. Statistical significance between young and aged (with and without Alzheimer's disease) was determined by MANOVA. Values at zero time were treated as covariate and time (10, 30 and 60 s) as a repeated measures factors. Sources of variation were nested as cell line within group, day within cell line, and experiments within day.

pholipid and on the intracellular calcium levels followed by nested analysis. For inositol phosphates, values at zero time were treated as covariates, and time (10, 30 and 60 s) as a repeated measures factor. Sources of variation were nested as cell line within group, day within cell line, and experiment within day. ANOVA followed by Student-Newman-Keul's test was also performed on the calcium levels and on the formation of inositol phosphates. The value for each inositol phosphate was expressed as a ratio to total phospholipid, and values at zero time were subtracted from each time point (10, 30 and 60 s). RESULTS The optimal incubation time for prelabeling the phosphoinositide with [3H]inositol in fibroblasts was determined previ-

471

ously (11). The amount of radiolabel incorporated into phosphoinositide (DPM/dish) varied between cell lines, but was similar to young, normal aged and Alzheimer donors. The temporal patterns and the dose responses of inositol phosphates or [Ca2+]i to bradykinin stimulation under these conditions have been characterized (11). The bradykinin-induced formation of IP 3 in fibroblasts from normal aged or Alzheimer patients was significantly higher than those from young donors. In all three groups, bradykinin induced a transient and rapid formation of IP 3, and the temporal pattern among the three groups appeared similar (Fig. 1A). If the normal aged and Alzheimer groups were compared separately to the young group, then bradykinin-induced IP 3 formation was significantly greater in fibroblasts from normal aged or Alzheimer donors than from young donors at 10 and 60 s [ANOVA 10 s, F(2,17)=3.7; 60 s, F(2,17)=4.3; p<0.05; Fig. 1A]. Between group analysis with the same analysis of variance demonstrated that no significant difference occurred between the normal aged and Alzheimer groups. If the values for the normal aged and Alzheimer donors were combined as one group, the magnitude of the response was elevated significantly in fibroblasts from old (with or without Alzheimer's disease) compared to that from young donors at all time points [MANOVA, F(1,15)=6.4, p=0.03]. Bradykinin stimulated formation of IP 2 linearly over the times observed, and the patterns of temporal responses appeared similar in the young, normal aged and Alzheimer groups (Fig. 1B). If normal aged and Alzheimer groups were compared separately to the young group, bradykinin-induced IP 2 formation was significantly greater in fibroblasts from normal aged or Alzheimer donors than from young at 60 s [ANOVA, F(2,17)=3.8; p<0.05; Fig. 1B]. If the normal aged and Alzheimer donors were combined as one group, the magnitude of the responses was increased significantly in aged (with or without Alzheimer's disease) donors compared to that in young donors at 60 s [MANOVA, F(1,15)= 12, p<0.01]. Bradykinin stimulated time-dependent changes in [Ca2+]i in fibroblasts, but this was not altered by aging or Alzheimer's disease (Fig. 2). Bradykinin increased [Ca2+]i from a basal level to a peak within 6-7 s, which was followed by a gradual decline over three minutes to a new equilibrium [see reference (11) for more detail]. [Ca2+]i was monitored continuously, but values at time points (0 s, peak time, 30 s, 60 s and 180 s) corresponding to those for the phosphoinositide cascade were calculated for statistical comparisons. Neither the basal levels of [Ca 2÷ ]~, nor the temporal patterns, nor the magnitude of [Ca2+] i in response to bradykinin at any time point differed significantly between young, normal aged or Alzheimer subjects [ANOVA, basal: F(2,17)=0.2; peak: F(2,17)=0.8; 30 s: F(2,17)=0.01; 60 s, F(2,17)=0.7; 180 s, F(2,17)=0.2; p>0.05]. DISCUSSION The stimulation of the phosphoinositide cascade by bradykinin was enhanced as the age of the donors increased. Aging elevated the bradykinin-induced formation of both IP 3 and IP 2. Since the calcium response did not change with the age of the donors, the age-related changes in the phosphoinositide cascade were not correlated with changes in [Ca2+] i. Bradykinin-activated changes in IP 3, normally associated with release of calcium from internal pools, do not necessarily correlate with intracellular calcium signals in human skin fibroblasts. Our previous studies demonstrate that with low concentrations of bradykinin, a submaximal IP 3 signal (<10% of full response) is associated with a full calcium response (11). This suggests that

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TIME (SECONDS) FIG. 2. Effects of bradykinin (100 nM) on the [Ca2+]i in cultured human skin fibroblasts from young, normal aged and Alzheimer donors. [Ca2+]i was estimated as described in the Method section. [Ca2+]i values at 0 (basal), 6-7 (peak time), 30, 60 and 180 s were calculated and compared statistically. Values are means--+SEM of six young, aged or Alzheimer cell lines. Each cell line was examined in quadruplicate on two different days. Significant differences (p<0.05) among groups were assessed by ANOVA followed by Student-Newman-Keul's test. Statistical significance between young and aged (with and without Alzheimer's disease) was tested by MANOVA. the smaller IP 3 signal in fibroblasts from young donors would still be sufficient to trigger a full calcium response. On the other hand, an increase in the bradykinin-induced phosphoinositide cascade by the aged donors may compensate for a decreased efficacy of IP 3 to elicit calcium mobilization with aging, as has been demonstrated in rat brain microsomes (4) and parotid cells (14). The age-related elevation in bradykinin-induced inositol polyphosphate generation may be the result of an increased number or affinity of receptors linked to phospholipase C, or to a more efficient coupling of receptor and GTP binding proteins. Although the physiological significance of these studies is unclear, in platelets, enhanced thrombin-induced phosphoinositide turnover with aging is correlated positively to aggregation activity (1). Further studies on these aspects of the signaling mechanisms will be necessary to ascertain the molecular basis of physiological impact of enhanced activation of phosphoinositide cascade with aging. The age-related increase of inositol polyphosphate formation after bradykinin suggests an age-related increase of diacylglycerol, which normally activates protein kinase C. Since activation of protein kinase C inhibits signal transduction systems by attenuating the phosphoinositide response (23), the age-related increase in the phosphoinositide cascade may indicate reduced protein kinase C activity. Prolonged activation of protein kinase C by phorbol ester is known to down regulate its activity (23). A similar effect with an enhanced phosphoinositide cascade would suggest this as a possible mechanism for a reduced pro-

tein kinase C activity in fibroblasts from Alzheimer donors. The total activity of protein kinase C is reduced in 24-month-old rat cerebral cortex (7), and the Concanavalin A-induced protein kinase C is reduced in T tymphocytes in 24-month-old mice (24). As mentioned previously, protein kinase C activity declines in brains (17) and fibroblasts (12) from Alzheimer patients. Whether the age-related changes in receptor-mediated phosphoinositide cascade correlate with protein kinase C activity in fibroblasts requires further study. The current results that demonstrate no change in fibroblast [Ca2÷]i with aging and Alzheimer's disease differ from two previous reports. Bradykinin induced a transient and rapid elevation of [Ca2+] i in fibroblasts from young, normal aged and Alzheimer donors. In the current studies, the pattern of the temporal responses, the magnitude of [Ca2+]t at basal or after bradykinin stimulation were similar among fibroblasts from young, normal aged and Alzheimer donors. The lack of change with Alzheimer's disease is not due to a ceiling effect. The concentration of bradykinin used is intermediate, so that both a stimulation and inhibition would have been apparent. Secondly, the temporal patterns of both [Ca 2÷ ]i and phospho-inositide cascade response to bradykinin would have readily revealed any ceiling effect, and none was apparent. However, previous reports suggest that basal and bradykinin- or serum-induced [Ca2÷]i are reduced in fibroblasts from young compared to normal aged donors, and further diminished in Alzheimer donors (21,22). On the other hand, another laboratory reports that serum-induced alterations in [Ca 2÷ ]i are exaggerated in fibroblasts from Alzheimer patients (26). It should be emphasized that the conditions in the current experiments were not designed to replicate the previous studies, but to examine the interactions of the phosphoinositide cascade and calcium with aging and Alzheimer's disease. Thus, the contrast between the current and previous results may, in part, be due to differences in cell seeding density, concentrations of serum during cell culture maintenance, degree of cell confluence, length of serum depletion, and whether single cell or multiple cells were examined. Although our studies did not detect a significant difference between young, normal aged or Alzheimer donors, this does not exclude the possibility that such a difference exits under certain cell culture conditions. Solid conclusions about the link between Ca 2+ homeostasis and the pathophysiology of aging and Alzheimer's disease cannot be drawn from the literature, including the current study with cultured fibroblasts. This does not mean that Alzheimer's disease or aging does not alter calcium in culture, but that the changes are sensitive to unknown factor(s) in tissue culture. Moreover, the results do demonstrate that the changes in calcium with aging or Alzheimer's disease are not so robust as to persist under a variety of conditions, even when another second messenger system (i.e., phosphoinositide cascade) is dramatically altered. However, the advantage of the cultured ceils is that pathophysiology may eventually be able to be examined in a controlled system. ACKNOWLEDGEMENT

The authors thank Dr. J. Appleby for his excellent assistance.

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