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Cyclic nucleotides in rabbit corneal endothelial cells: Fresh tissue vs. tissue culture
Exp. Eye Res. (1979) 28, 285-289
Cyclic Nucleotides in Rabbit Cornea1 Endothelial Cells: Fresh Tissue vs. Tissue Culture R. THEODORE
DAVID R. WHIKEHARTAND
FLETCHER
Laboratory of Vision Research,National Eye Institute, Bethesda,Md. 20014, l!.S.A. (Received3 August 1978, New York) Quantities of adenosine 3’,5’-cyclic monophosphate were measured in rabbit cornea1 endothelial cells taken from fresh tissue and tissue culture. Higher levels of both nucleotides were observed when the cells were initially frozen in liquid nitrogen compared with those that were not. Adenosine 3’,5’-cyclic monophosphate was 14 times lower in the primary cultures compared to the fresh tissues while subcultures were about two-thirds that of the fresh tissue amount. Guanosine 3’,5’-cyclic monophosphate was four times higher in the primary cultures compared to the fresh tissue while subcultures were about two-thirds that of the fresh tissue amount. It is emphasized that the ratios of adenosine 3’,5’-cyclic monophosphate to guanosine 3’,5’-cyclic monophosphate were similarly high in both fresh tissue and subcultured cells approaching confluency, but quite low in primary outgrowths. Key zuords: adenosine 3’,5’-cyclic monophosphate; guanosine 3’,5’-cyclic monophosphate: cornea: endothelium ; tissue culture.
1. Introduction The effects of adenosine 3’,5’-cyclic monophosphate (CAMP) and guanosine 3’,5’cyclic monophosphate (cGMP) on cell growth and other functions have beenvigorously pursued in recent years (Paston, Johnson and Anderson, 1975; Goldberg and Ha&lox, 1977). However, in the cornea1 endothelium such investigations have been sparse and discouraging. Neufeld and Sears (1974) reported the absenceof any CAMP in an endothelial fraction of monkey cornea. Moreover, Dikstein (1973) had previously described the lack of any effect of adenylate cyclase (ATP pyrophosphate lyase (cyclizing) 4.6.1.1) as a system in the operation of the endothelial pump. There have been apparently no studies of cGMP in this tissue. The purpose of this investigation, therefore, is a thorough attempt to ascertain whether there might be measurable quantities of both CAMP and cGMP in the cornea1 endothelial cell. It would further attempt to compare measurable levels of these cyclic nucleotides in fresh tissue with those present in tissue cultures. Values from tissue cultures could accordingly serve as a basis for the comparison of the biochemical similarity of these cells with those present in the fresh tissue. 2. Methods New Zealand white rabbits
(1000 g) were used as a source of both fresh tissue and for the
initiation of tissue cultures. All rabbits were sacrificed by carbon dioxide intoxication. Fresh tissue was prepared
for assay in two ways. In the first method,
the scrapings from
four endothelia per assay were placed in either 1.2 ml 6% TCA or 10% PCA (ice bath) and homogenized Reprint Birmingham,
for 1 min. In the second method,
requests: Dr Birmingham,
0014-4836/79/030285+05
David R. Alabama
Whikehart, School 35294, U.S.A.
four cornea1 buttons of
Optometry,
0 1979 Academic
$01.00/0 285
University
per assay were of Alabama
Press Inc. (London)
Limited
in
2%
D.
It.
\VHIKEHAfiT
ASl,
R.. ‘1’. FLETCHJSK
placed in a 35 mm petri dish. Dish and contents were cooled in liquid nitrogen an11 I.3 ml of TCA or PCA (as above) were added and quickly swirled to clJver the eutire dish ;tntl contents before freezing. The extra 100 ~1 was found to be more convenient for processing. The endothelial layer was scraped loose after allowing the TCA (or PCA) to warn1 up to an ice water stage and the suspensions was transferred t,o a homogenizer and homogenized (ic,e bath) for 1 min. Aliquots of 20 ~1 were removed from each assay preparation for protein determination (Lees and Paxman, 1972). Tissue cultures were grown essentially by the method of Perlman and Baum (1974). All tissue culture medium components, except antibiotics, were obtained from Microbiological Associates. The original medium was modified by the inclusion of 7.30 pg/tnl sodium bicarbonate, 10 pg/ml Gentamicin (Schering Co.. supplied by Microbiological Associates) and 2*5yg/ml Fungizone (Amphotericin B, R. Squibb & Sons, supplied b) Grand Island Biological Company) as recommended by Baum (private communication). The primary outgrowths were either harvested for assay or subcultured after ‘i-10 days. Subcultured cells were allowed to grow toward confluency (7-10 days). Tissue cultures were prepared for assay in two ways. In the first method, the medium from each petri dish was decanted and the cells were washed x 2 with minimum essential medium (Eagle). Cold 6% TCA or 10% PCA (1.3 ml) was added to each dish and the cells were scrape(l loose. Generally, the scrapings from two dishes were combined in the original 1.3 ml volume. The suspensions were homogenized in the cold for 1 min. Primary outgrowths were not assayed by the first method. In the second method, the medium was removed and cells were washed as before. Primary cultures had their buttons carefully removed. Each dish and contents were cooled in liquid nitrogen followed by the addition of eit#her TCA or PCA (1.3 ml) as in the first method with swirling to allow the TCA (or PCA) to cover the entire dish before freezing. Scrapings were made, after allowing the TCA (or PCA) to reach an ice water stage, but were not combined. The suspensions were hornogenized as above. Twenty ~1 were removed from each suspension for protein assay. Sssays for CAMP and cGMP were performed essentially by the method of Steiner, Parker and Kipnis (1972) with reagents supplied by New England Nuclear. Cyclic: nucleotides were isolated from SO-120 pg protein equivalents of tiupernatants (2501) >; g, 15 min) from tissue suspensions (250-350 pg of protein) by passing each through a column of either Dowex BOW-X8 (H+) using water for elution (Steiner, 1974) or Biorad AG l-S8 (formate) using formic acid for elution (Krishnan and Krishna, 1976). Sample recoveries of the cyclic nucleotides were in the range of 8( l-90q,0 with either column.
3. Results In the initial
attempt
to measure
CAMP
and cGMP,
homogenates
were prepared
from cells that were scraped from their surrounding tissue at room temperature. The amounts of cyclic nucleotides found in these cells is shown in Table I (under “RT”). The amounts of CAMP in both fresh tissue cells and cells grown in culture (approaching the confluent stage after subculturing) were nearly equivalent (U > 0.05). Levels of cGMP were considerably lower than CAMP as shown. The amount of cGMP, however, in cultured cells was significantly lower (P <: O-05)than the cGMP of fresh tissue cells. A refinement, of the process of measuring CAMP and cGMP consisted of quickly freezing the cornea1endothelial cells before scraping in order to prevent metabolic changes that might significantly alter the levels. Table I (under “NZ”) indicates the same pattern of amounts of cyclic nucleotides, but at considerably elevated levels compared with the results of the earlier technique. This was especially marked for CAMP.
The remaining
results
nitrogen quick freeze method.
are reported
for tissue
samples
done
by the liquid
CYCLIC’
NUCLEOTIDES
IN
RABBIT
CORNEAL
ENDOTHELIAL
287
CELLS
Cyclic AMP was measured in primary outgrowths of cornea1 endothelial cells as well as from fresh tissue and subcultured cells approaching confluency. This is indicat~ed in Table II. Note that the CAMP was considerably lower (approximat.ely 14 times) in the primary culture than in the fresh tissue cells. In cells approaching confluency (after subculturing) CAMP was about two-thirds that of the fresh tissue amount. The difference of the fresh tissue cells and the cells in subculture was statistically significant (0.05 > P > O-02). TABLE
I
Cyclic nu,cleotides: room temperflture vs. l~iquid nitrogen processing*
8ample Fresh cells (fultured cell&j * Average S Subcultured,
value in pmol/mg protein; approaching confluency.
8.6 7.0
I.8 0.4
t Room
temperature
TABLE
2.3 1,s
60.7 4133 processing;
$. Liquid
nitrogen
processing:
II
Cyclic nucleotides: comparison of amouds ivb jyrimary cultures with fresh cells and subcultures*
Sample
Fresh cells Primary culture Subculturei * After
liquid
nitrogen
treatment;
CAMP (pmol/mg
60.7k4.1 4.21-0.4 41+Q&7 t 3lean~S.E.
(8)t (6) (5)
CGMP protein)
L?+J*o~L) (4) 11.X&0.6 (i) 1%&O? (8)
(R determinat~ions);
22 0.4 2: 3
$ (Jells approaching
confluency.
Cyclic GMP levels are also shown in Table II for fresh tissue cells and cells from primary and subcultured growths. Note that the cGMP was considerably higher (approximately four times) in primary culture than in the fresh tissue cells. Suhcultured cells, approaching confluency, have a cGMP level close to the fresh tissue cell amount. The difference of the fresh tissue cells and the cells in subculture were, however, significantly different (P < 0.01). Ratios of cAMP/cGMP are indicated in Table II. Both fresh tissue cells and subcultured, nearly confluent cells had high and nearly identical ratios. Primary cultures, however, had cells with very low ratios (about 55 times lower) compared to the other cells. 4. Discussion The determination of high amounts of CAMP and cGMP in cornea1 endothelial cells (both fresh and from culture) may indicate their importance in this tissue. Cyclic AMP concentrations in rat tissues, for example, in pmol/mg protein, are 0*097-0.144
.%s
1). R,. WHIKEHART
AND
R.
‘1’. FLETCHER
(liver), O-018-0.087 (skeletal muscle), and 0*005-0.012 (fat cells) whereas c(+RlP concentrations are O-002 (liver), 0*00220*004 (skeletal muscle), and 0~0003-0~0007 (fat cells) (Steiner et al., 1972). In this study a, value of 0.09i pm01cAMP/mg protein was obtained for mouse liver by processing the tissue without liquid nitrogen. lu ocular tissues some CAMP representative levels are (in pmol/mg protein): 1.X (dark adapted frog rod outer segments), 3.4 (light adapted frog rod outer segments) (Fletcher and Chader, 1976), 16 (rabbit whole cornea), 13 (rabbit iris-ciliary body) and 20 (retina-choroid) (Neufeld, Jampol and Sears, 19i2). Corresponding cGMP values (pmol/mg protein) are : 29.3 (dark adapted frog rod outer segments).3.1 (light adapted frog outer segments) (Fletcher and Chader, 1976), O-042 (rabbit lens), 0.46 (rabbit iris-ciliary body) and 4.94 (choroid) (B onomi, Fregona and Tomazzoli. 1977). Although concentrations of the order of 61 pm01cAMP/mg protein seemhigh (cornea1 endothelium, this study), it is known that levels may become greater than 600 pmol/ mg protein for cGMP in neuroblastoma cells (Matsuzawa and Nirenberg, 1975) upon activation of muscarinic acetylcholine receptors. It is customary to suspect that prostaglandins may rapidly stimulate the production of high levels of CAMP I)! activating adenylate cyclase (Paston, Johnson and Anderson, 1975; Samuelxson, Granstrom, Green, Hamberg and Hammarstrom, 1975) as a result of cell scraping at room temperature. Consequently, it was unexpected that treatment with liquid nitrogen resulted in higher CAMP levels (Table I). However, prostaglandins are also known to have inhibitory effects in some instances (fat cells. stomach and kidney cortex tissues) (Samuelssonet al., 1975). It is not certain whether prostaglandins have any effects on cGMP (Goldberg and Haddox, 1977) although the results of this study indicated a small and significant (P < 0.05) inhibitory effect. The ratios of CAMP to cGMP (Table II) are in the normal range in the rabbit cornea1endothelial cells in fresh tissue and in cultured cells approaching confluency, when compared with most tissues (Kolata, 1973). However, it is interesting to note the reversal of ratios and levels of both CAMP and cGMP going from fresh tissue t,o primary outgrowths to subcultured cells approaching confluency (Table II). The effect is a classicalexample of Goldberg’s yin-yang hypothesis (Goldberg, O’Dea and Haddox, 1973) in which cGMP is elevated relative to CAMP during rapid growth. Do the cyclic nucleotides present in these cells have any effect on the deturgescent pump mechanism? It is known that the activity of guanylate cyclase is affected by alterations to its redox state (Goldberg and Haddox, 1977). Reduced glutathione and cysteine.. for example, prevent activation while hydrogen peroxide promotes it. Dehydroascorbate activates guanylate cyclase while inhibiting adenylate cyclase. Adenosine, in the specific instance of neuroblastoma cells, can raise CAMP levels 500% while lowering cGMP to 407; of its original value (Matsuzawa and Nirenberg! 1975). Someof these agents have been implicated in deturgescent activation (Dikstein and Maurice, 1972; Anderson, Fischbarg and Spector, 1974; Whikehart and Edelhauser, 1978). Moreover, the concentration of ascorbic acid in the bovine cornea1 endothelium is normally quite high (761 pg/g tissue wet wt.) (Whikehart, unpublished observation) and could contribute to the phenomenon. The exact role of the cyclic nucleotides, however, is unknown.
ACKNOWLEDGMENTS
The authors
would like to acknowledge
the helpful
advice of Dr Gerald Chader, National
Eye Institute, and Dr Jules Baum, New England 81edicalCenter.
CYCLIC
NUCLEOTIDES
IN
RABBIT
CORNEAL
ENDOTHELIAL
CELLS
289
REFERENCES Anderson, E. I., Fischbarg, J. and Spector, A. (1974). Disulfide stimulation of fluid transport and effect on ATP level in rabbit cornea1 endothelium. Eye Exp. Res. 19, l-10. monophosphate (GMP) levels Bonomi, L., Fregona, J. and Tomazolli, L. (1977). Cy c1ic guanosine in ocular tissues. Albrecht von Gruqfes Arch. R&n. Exp. Ophthalmol. 205,23-7. Dikstein, 8. (1973). Efficiency and survival of the cornea1 endothelial pump. Exp. Eye Res. 15,
63944. Dikstein, S. and Maurice, D. M. (1972). The metabolic basis for the fluid pump in the cornea. J. Physiol. (London) 221,2941. Fletcher, R. T. and Chader, G. J. (1976). Cyclic GMP: control of concentration by light in ret’inal photoreceptors. Biochem. Biophys. Acta 70, 1297-302. Goldberg, N. D., O’Dea, R. F. and Haddox, M. K. (1973). Cylic GMP. Adz?. Cyclic iV&eotide Res. 3, 155-3. Kolata, G. B. (1973). Cyclic GMP: cellular regulatory agent? Science 182, 14961. Krishnan, N. and Krishna, G. (1976). A simple and sensitive assay for guanylate cyclase. AmcI.$. Biochem. 70,18-31. Lees, M. B. and Paxman, S. (1972). Modification of the Lowry procedure for assays of proteolipid protein. Analyt. Biochem. 47, 184-92. Matsuzawa, H. and Nirenberg, M. (1975). Receptor-mediated shifts in cGMP and CAMP levels in neuroblastoma cells. Biochemistry 72,3472-6. Neufeld, A. H., Jampol, L. M. and Sears, M. L. (1972). Cyclic-AMP in the aqueous humor: the effects of adrenergic agents. Exp. Eye Res. 14,242-50. Neufeld, A. H. and Sears, M. L. (1974). Cylic-AMP in ocular tissues of the rabbit, monkey and human. Invest. Ophthalmol. 13,475-7. Paston, I. H., Johnson. G. S. and Anderson, W. 3. (1975). Role of cyclic nucleotides in growth control. Ann. Rev. Biochem. #, 491-522. Perlman, M. and Baum, J. L. (1974). The mass culture of rabbit cornea1 endothelial cells. A,rch. Ophthalmol. 92, 235-7. Samuelsson, B., Granstrom, E., Green, K., Hamberg. M. and Hammarstrom, S. (1975). Prostaglandins. Ana. Rev. Biochem. 44, 669495. Steiner, ,4. L. (1974). Assay of cyclic nucleotides. In llfethods in. Enzynwlogy (Eds Hardman, J. G. and O’Malley, B. W.). Vol. 38, pp. 96-105. Academic Press, N.Y. Steiner, A. L., Parker, C. W. and Kipnis, D. M. (1972). Radioimmunoassay for cyclic nucleotides. J. Biol. Chem. 247, 1106-13. Steiner, A. L., Wehmann, R. E., Parker, C. W. and Kipnis, D. M. (1972). Radioimmunoassay for the measurement of cyclic nucleotides. A&J. Cyclic Nucleotide Res. 2, 51-61. Whikehart, D. R. and Edelhauser, H. F. (1978). Glutathione in rabbit cornea1 endothelia: the effects of selected perfusion fluids. Invest. Ophthulmol. Vis. Sci. 17, 455-64.
Cyclic Nucleotides in Rabbit Cornea1 Endothelial Cells: Fresh Tissue vs. Tissue Culture R. THEODORE
DAVID R. WHIKEHARTAND
FLETCHER
Laboratory of Vision Research,National Eye Institute, Bethesda,Md. 20014, l!.S.A. (Received3 August 1978, New York) Quantities of adenosine 3’,5’-cyclic monophosphate were measured in rabbit cornea1 endothelial cells taken from fresh tissue and tissue culture. Higher levels of both nucleotides were observed when the cells were initially frozen in liquid nitrogen compared with those that were not. Adenosine 3’,5’-cyclic monophosphate was 14 times lower in the primary cultures compared to the fresh tissues while subcultures were about two-thirds that of the fresh tissue amount. Guanosine 3’,5’-cyclic monophosphate was four times higher in the primary cultures compared to the fresh tissue while subcultures were about two-thirds that of the fresh tissue amount. It is emphasized that the ratios of adenosine 3’,5’-cyclic monophosphate to guanosine 3’,5’-cyclic monophosphate were similarly high in both fresh tissue and subcultured cells approaching confluency, but quite low in primary outgrowths. Key zuords: adenosine 3’,5’-cyclic monophosphate; guanosine 3’,5’-cyclic monophosphate: cornea: endothelium ; tissue culture.
1. Introduction The effects of adenosine 3’,5’-cyclic monophosphate (CAMP) and guanosine 3’,5’cyclic monophosphate (cGMP) on cell growth and other functions have beenvigorously pursued in recent years (Paston, Johnson and Anderson, 1975; Goldberg and Ha&lox, 1977). However, in the cornea1 endothelium such investigations have been sparse and discouraging. Neufeld and Sears (1974) reported the absenceof any CAMP in an endothelial fraction of monkey cornea. Moreover, Dikstein (1973) had previously described the lack of any effect of adenylate cyclase (ATP pyrophosphate lyase (cyclizing) 4.6.1.1) as a system in the operation of the endothelial pump. There have been apparently no studies of cGMP in this tissue. The purpose of this investigation, therefore, is a thorough attempt to ascertain whether there might be measurable quantities of both CAMP and cGMP in the cornea1 endothelial cell. It would further attempt to compare measurable levels of these cyclic nucleotides in fresh tissue with those present in tissue cultures. Values from tissue cultures could accordingly serve as a basis for the comparison of the biochemical similarity of these cells with those present in the fresh tissue. 2. Methods New Zealand white rabbits
(1000 g) were used as a source of both fresh tissue and for the
initiation of tissue cultures. All rabbits were sacrificed by carbon dioxide intoxication. Fresh tissue was prepared
for assay in two ways. In the first method,
the scrapings from
four endothelia per assay were placed in either 1.2 ml 6% TCA or 10% PCA (ice bath) and homogenized Reprint Birmingham,
for 1 min. In the second method,
requests: Dr Birmingham,
0014-4836/79/030285+05
David R. Alabama
Whikehart, School 35294, U.S.A.
four cornea1 buttons of
Optometry,
0 1979 Academic
$01.00/0 285
University
per assay were of Alabama
Press Inc. (London)
Limited
in
2%
D.
It.
\VHIKEHAfiT
ASl,
R.. ‘1’. FLETCHJSK
placed in a 35 mm petri dish. Dish and contents were cooled in liquid nitrogen an11 I.3 ml of TCA or PCA (as above) were added and quickly swirled to clJver the eutire dish ;tntl contents before freezing. The extra 100 ~1 was found to be more convenient for processing. The endothelial layer was scraped loose after allowing the TCA (or PCA) to warn1 up to an ice water stage and the suspensions was transferred t,o a homogenizer and homogenized (ic,e bath) for 1 min. Aliquots of 20 ~1 were removed from each assay preparation for protein determination (Lees and Paxman, 1972). Tissue cultures were grown essentially by the method of Perlman and Baum (1974). All tissue culture medium components, except antibiotics, were obtained from Microbiological Associates. The original medium was modified by the inclusion of 7.30 pg/tnl sodium bicarbonate, 10 pg/ml Gentamicin (Schering Co.. supplied by Microbiological Associates) and 2*5yg/ml Fungizone (Amphotericin B, R. Squibb & Sons, supplied b) Grand Island Biological Company) as recommended by Baum (private communication). The primary outgrowths were either harvested for assay or subcultured after ‘i-10 days. Subcultured cells were allowed to grow toward confluency (7-10 days). Tissue cultures were prepared for assay in two ways. In the first method, the medium from each petri dish was decanted and the cells were washed x 2 with minimum essential medium (Eagle). Cold 6% TCA or 10% PCA (1.3 ml) was added to each dish and the cells were scrape(l loose. Generally, the scrapings from two dishes were combined in the original 1.3 ml volume. The suspensions were homogenized in the cold for 1 min. Primary outgrowths were not assayed by the first method. In the second method, the medium was removed and cells were washed as before. Primary cultures had their buttons carefully removed. Each dish and contents were cooled in liquid nitrogen followed by the addition of eit#her TCA or PCA (1.3 ml) as in the first method with swirling to allow the TCA (or PCA) to cover the entire dish before freezing. Scrapings were made, after allowing the TCA (or PCA) to reach an ice water stage, but were not combined. The suspensions were hornogenized as above. Twenty ~1 were removed from each suspension for protein assay. Sssays for CAMP and cGMP were performed essentially by the method of Steiner, Parker and Kipnis (1972) with reagents supplied by New England Nuclear. Cyclic: nucleotides were isolated from SO-120 pg protein equivalents of tiupernatants (2501) >; g, 15 min) from tissue suspensions (250-350 pg of protein) by passing each through a column of either Dowex BOW-X8 (H+) using water for elution (Steiner, 1974) or Biorad AG l-S8 (formate) using formic acid for elution (Krishnan and Krishna, 1976). Sample recoveries of the cyclic nucleotides were in the range of 8( l-90q,0 with either column.
3. Results In the initial
attempt
to measure
CAMP
and cGMP,
homogenates
were prepared
from cells that were scraped from their surrounding tissue at room temperature. The amounts of cyclic nucleotides found in these cells is shown in Table I (under “RT”). The amounts of CAMP in both fresh tissue cells and cells grown in culture (approaching the confluent stage after subculturing) were nearly equivalent (U > 0.05). Levels of cGMP were considerably lower than CAMP as shown. The amount of cGMP, however, in cultured cells was significantly lower (P <: O-05)than the cGMP of fresh tissue cells. A refinement, of the process of measuring CAMP and cGMP consisted of quickly freezing the cornea1endothelial cells before scraping in order to prevent metabolic changes that might significantly alter the levels. Table I (under “NZ”) indicates the same pattern of amounts of cyclic nucleotides, but at considerably elevated levels compared with the results of the earlier technique. This was especially marked for CAMP.
The remaining
results
nitrogen quick freeze method.
are reported
for tissue
samples
done
by the liquid
CYCLIC’
NUCLEOTIDES
IN
RABBIT
CORNEAL
ENDOTHELIAL
287
CELLS
Cyclic AMP was measured in primary outgrowths of cornea1 endothelial cells as well as from fresh tissue and subcultured cells approaching confluency. This is indicat~ed in Table II. Note that the CAMP was considerably lower (approximat.ely 14 times) in the primary culture than in the fresh tissue cells. In cells approaching confluency (after subculturing) CAMP was about two-thirds that of the fresh tissue amount. The difference of the fresh tissue cells and the cells in subculture was statistically significant (0.05 > P > O-02). TABLE
I
Cyclic nu,cleotides: room temperflture vs. l~iquid nitrogen processing*
8ample Fresh cells (fultured cell&j * Average S Subcultured,
value in pmol/mg protein; approaching confluency.
8.6 7.0
I.8 0.4
t Room
temperature
TABLE
2.3 1,s
60.7 4133 processing;
$. Liquid
nitrogen
processing:
II
Cyclic nucleotides: comparison of amouds ivb jyrimary cultures with fresh cells and subcultures*
Sample
Fresh cells Primary culture Subculturei * After
liquid
nitrogen
treatment;
CAMP (pmol/mg
60.7k4.1 4.21-0.4 41+Q&7 t 3lean~S.E.
(8)t (6) (5)
CGMP protein)
L?+J*o~L) (4) 11.X&0.6 (i) 1%&O? (8)
(R determinat~ions);
22 0.4 2: 3
$ (Jells approaching
confluency.
Cyclic GMP levels are also shown in Table II for fresh tissue cells and cells from primary and subcultured growths. Note that the cGMP was considerably higher (approximately four times) in primary culture than in the fresh tissue cells. Suhcultured cells, approaching confluency, have a cGMP level close to the fresh tissue cell amount. The difference of the fresh tissue cells and the cells in subculture were, however, significantly different (P < 0.01). Ratios of cAMP/cGMP are indicated in Table II. Both fresh tissue cells and subcultured, nearly confluent cells had high and nearly identical ratios. Primary cultures, however, had cells with very low ratios (about 55 times lower) compared to the other cells. 4. Discussion The determination of high amounts of CAMP and cGMP in cornea1 endothelial cells (both fresh and from culture) may indicate their importance in this tissue. Cyclic AMP concentrations in rat tissues, for example, in pmol/mg protein, are 0*097-0.144
.%s
1). R,. WHIKEHART
AND
R.
‘1’. FLETCHER
(liver), O-018-0.087 (skeletal muscle), and 0*005-0.012 (fat cells) whereas c(+RlP concentrations are O-002 (liver), 0*00220*004 (skeletal muscle), and 0~0003-0~0007 (fat cells) (Steiner et al., 1972). In this study a, value of 0.09i pm01cAMP/mg protein was obtained for mouse liver by processing the tissue without liquid nitrogen. lu ocular tissues some CAMP representative levels are (in pmol/mg protein): 1.X (dark adapted frog rod outer segments), 3.4 (light adapted frog rod outer segments) (Fletcher and Chader, 1976), 16 (rabbit whole cornea), 13 (rabbit iris-ciliary body) and 20 (retina-choroid) (Neufeld, Jampol and Sears, 19i2). Corresponding cGMP values (pmol/mg protein) are : 29.3 (dark adapted frog rod outer segments).3.1 (light adapted frog outer segments) (Fletcher and Chader, 1976), O-042 (rabbit lens), 0.46 (rabbit iris-ciliary body) and 4.94 (choroid) (B onomi, Fregona and Tomazzoli. 1977). Although concentrations of the order of 61 pm01cAMP/mg protein seemhigh (cornea1 endothelium, this study), it is known that levels may become greater than 600 pmol/ mg protein for cGMP in neuroblastoma cells (Matsuzawa and Nirenberg, 1975) upon activation of muscarinic acetylcholine receptors. It is customary to suspect that prostaglandins may rapidly stimulate the production of high levels of CAMP I)! activating adenylate cyclase (Paston, Johnson and Anderson, 1975; Samuelxson, Granstrom, Green, Hamberg and Hammarstrom, 1975) as a result of cell scraping at room temperature. Consequently, it was unexpected that treatment with liquid nitrogen resulted in higher CAMP levels (Table I). However, prostaglandins are also known to have inhibitory effects in some instances (fat cells. stomach and kidney cortex tissues) (Samuelssonet al., 1975). It is not certain whether prostaglandins have any effects on cGMP (Goldberg and Haddox, 1977) although the results of this study indicated a small and significant (P < 0.05) inhibitory effect. The ratios of CAMP to cGMP (Table II) are in the normal range in the rabbit cornea1endothelial cells in fresh tissue and in cultured cells approaching confluency, when compared with most tissues (Kolata, 1973). However, it is interesting to note the reversal of ratios and levels of both CAMP and cGMP going from fresh tissue t,o primary outgrowths to subcultured cells approaching confluency (Table II). The effect is a classicalexample of Goldberg’s yin-yang hypothesis (Goldberg, O’Dea and Haddox, 1973) in which cGMP is elevated relative to CAMP during rapid growth. Do the cyclic nucleotides present in these cells have any effect on the deturgescent pump mechanism? It is known that the activity of guanylate cyclase is affected by alterations to its redox state (Goldberg and Haddox, 1977). Reduced glutathione and cysteine.. for example, prevent activation while hydrogen peroxide promotes it. Dehydroascorbate activates guanylate cyclase while inhibiting adenylate cyclase. Adenosine, in the specific instance of neuroblastoma cells, can raise CAMP levels 500% while lowering cGMP to 407; of its original value (Matsuzawa and Nirenberg! 1975). Someof these agents have been implicated in deturgescent activation (Dikstein and Maurice, 1972; Anderson, Fischbarg and Spector, 1974; Whikehart and Edelhauser, 1978). Moreover, the concentration of ascorbic acid in the bovine cornea1 endothelium is normally quite high (761 pg/g tissue wet wt.) (Whikehart, unpublished observation) and could contribute to the phenomenon. The exact role of the cyclic nucleotides, however, is unknown.
ACKNOWLEDGMENTS
The authors
would like to acknowledge
the helpful
advice of Dr Gerald Chader, National
Eye Institute, and Dr Jules Baum, New England 81edicalCenter.
CYCLIC
NUCLEOTIDES
IN
RABBIT
CORNEAL
ENDOTHELIAL
CELLS
289
REFERENCES Anderson, E. I., Fischbarg, J. and Spector, A. (1974). Disulfide stimulation of fluid transport and effect on ATP level in rabbit cornea1 endothelium. Eye Exp. Res. 19, l-10. monophosphate (GMP) levels Bonomi, L., Fregona, J. and Tomazolli, L. (1977). Cy c1ic guanosine in ocular tissues. Albrecht von Gruqfes Arch. R&n. Exp. Ophthalmol. 205,23-7. Dikstein, 8. (1973). Efficiency and survival of the cornea1 endothelial pump. Exp. Eye Res. 15,
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