Effects of isoproterenol and cyclic AMP derivatives on cell division in cultured rat lenses. Ludwig von Sallmann and Patricia Grimes

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1 Effects of isoproterenol and cyclic AMP derivatives on cell division in cultured rat lenses Ludwig von Sallmann and Patricia Grimes Changes in proliferative activity in epithelial cells of rat lenses maintained in organ culture for 24 hours have been characterized, and the effects of isoproterenol (IPR) and cyclic adenosine-3',5'-monophosphate(camp) derivatives added to the culture medium have been studied. Transient mitotic inhibition occurred within the first few hours of incubation with IPR as well as with monobutyryl and dibutyryl camp. Propranolol blocked the action of IPR. Suppression of cell division was initiated in the G t period of the cell cycle without any indication of a d inhibition. The results of these experiments demonstrate that stimulation of beta receptors in the isolated lens by IPR and elevation of camp levels can directly affect cell proliferation in the epithelium. Key words: Lens organ culture, rat lens epithelium, cell division, isoproterenol, propranolol, dibutyryl cyclic AMP, monobutyryl cyclic AMP, butyrate. M any recent studies have indicated that cell division and differentiation in certain mammalian cells are influenced through hormonal stimulation of the adenylate cyclase system by a variety of agents, including beta-adrenergic agonists. We have reported that isoproterenol (IPR), a synthetic analog of the natural catecholamines, inhibits cell division in the lens epithelium of living rats after either systemic or local administration. 1 The depression of cell division appeared to be From the Laboratory of Neurophysiology, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Md Manuscript submitted for publication Sept. 14, 1973; manuscript accepted for publication Nov. 9, caused by an event occurring in the G] phase of the cell cycle which induced a transient fall in the number of cells synthesizing deoxyribonucleic acid (DNA), followed by a consequent reduction in mitotic activity. The beta-adrenergic blocking agent, propranolol, completely eliminated IPR-induced mitotic inhibition. Furthermore, potentiation of the IPR effect by theophylline pointed to cyclic adenosine-3', 5'-monophosphate (camp) as a mediator of the response in the lens epithelium.- Makman and Kern 3 have demonstrated the presence of catecholamine-stimulated adenylate cyclase activity in rat lens, particularly in the combined capsuleepithelium portion. Our evidence suggested that the response to IPR resulted from stimulation of receptor sites within the eye, but the ex-

2 Volume 13 Number 3 Effects of IPR and cyclic AMP on cell division 211 Table I. DNA synthesis and mitosis in epithelium of rat lenses during 24 hours in culture Uncultured control lenses: Cultured lenses: 2 hours 4 hours 6 hours 8 hours 16 hours 24 hours Mean ± (20) 247 Mitoses S.E. ± 7 (62) 96 ± 4 (107) 166 ± 4 (18) 168 ± 10 (17) 149 ± 6 (36) 135 ± 6 (203) 109 ± 2 Per cent control 39" 67' The figures in parentheses represent the number of preparations in each group. "Difference from control value is significant at a probability level of p < H 3 -labeled Mean ± S. E. (20) 1,907 ± 60 (13) 2,083 ± (11) 1,942 ± (12) 1,558 ± (12) 1,182 ± cells Per cent control periments did not establish whether the lens itself was the target tissue for the catecholamine. In the present study, we have tested the effects of IPR on the isolated lens maintained in organ culture to determine if the drug acts directly on the lens in modifying cell division. Moreover, we have examined the influence of camp derivatives on cell proliferation under the same conditions. Methods Lenses from 160 to 180 gram Osborne-Mendel rats (approximately 6 weeks of age) were used in all experiments. The animals were killed with chloroform. The eyes were removed rapidly and immersed in warmed culture medium rapidly and Under 10x magnification, the eye was opened posteriorly by severing the optic nerve at its entrance to the globe. From this foramen, Vannas scissors were used to cut through the sclera, choroid, and retina along one meridian forward to the limbus. The incision was extended perilimbally in a plane through the ciliary processes, the root of the iris, and the peripheral cornea. The lens was then gently rolled free of the adherent tissue. Great care was taken to prevent the dissecting instruments from touching the lens. Each lens was immediately transferred to a small culture dish (Falcon, 35 by 10 mm.) containing 4 ml. of medium, held within a larger dish (Falcon, 60 by 15 mm.). The culture medium employed was that recommended by Kinoshita (personal communication) for incubation of the rat lens. It is a mixture of 50 parts of TC199, 20.5 parts of bicarbonate buffer, 4 and 9.5 parts of a solution containing sufficient glucose, fructose, and calcium chloride to achieve final concentrations of 5 mm, 30 mm, and 1.5 mm, respectively. 5 The lenses were incubated in an atmosphere of 95 per cent air and 5 per cent carbon dioxide at a temperature of 36.5 C. The ph of the medium under these conditions was DL-isoproterenol HC1 and theophylline were obtained from Schwartz-Mann, Orangeburg, N. Y.; N, O 2 '-dibutyryl adenosine-3',5'-monophosphate (DBcAMP) and N (! -monobutyryl adenosine-3',5'- monophosphate (MBcAMP) from Sigma Chemical Co., St. Louis, Mo.; and DL-propranolol HC1 from Ayerst Laboratories, New York, N. Y. The agents were freshly dissolved in the culture medium before the start of each experiment. Butyric acid (Sigma) was neutralized with sodium hydroxide before it was added to the medium. At the end of the incubation period the lenses were examined for clarity with a dissecting microscope and fixed in ethanol-acetic acid. Feulgenstained whole mounts of the epithelium were prepared and the number of mitoses per preparation was counted. To study DNA synthesis, H H - thymidine (New England Nuclear Corporation, Boston, Mass., 6.7 Ci. per millimole) was added to the culture medium in a concentration of 3 fid per milliliter one hour prior to fixation of the lenses, and radioautographs of the epithelial preparations were made. Results Cell proliferation in lenses incubated in control medium. DNA synthesis and mitosis had not been previously studied in the cultured rat lens. We, therefore, averaged all data from lenses incubated in control medium to provide the baseline against which effects of various test agents were measured. The numbers of mitoses and of cells incorporating H 3 -thymidine at intervals during the first 24 hours of culture are listed in Table I. These figures are also expressed

3 212 von Sallmann and Grimes Investigatioe Ophthalmology March 1974 Fig. 1. Effect of culture on cells of the meridional rows. This region of the lens epithelium has a normal appearance at four hours of incubation (A) but at 24 hours (B) shows extensive nuclear disintegration and disorganization. (Feulgen stain, x350.) as per cent of the average values obtained in uncultured lenses of normal rats of the same age. During the period of culture, rpthymidine incorporation and mitosis were disassociated and the mitotic process was inhibited more severely than DNA synthesis. The number of HMabeled cells was not significantly less than normal until 16 hours of isolation, in contrast to cell division which fell abruptly in the first two hours after transfer to culture. The relatively greater suppression of mitosis in comparison with DNA synthesis persisted throughout the 24 hour incubation. The epithelium maintained its morphologic organization without signs of cell death except in the meridional rows, The cells of this region underwent progressive destruction beginning between six and eight hours and continuing to 24 hours. By 24 hours the rows were shorter, were disarranged, and contained many remnants of fragmented nuclei (Fig. 1). Rat lenses kept in culture for longer than 24 hours showed a pattern of gradual disorganization of the epithelium. Mitotic activity increased in the pre-equatorial and central regions by 48 hours, peaked be- tween 48 and 72 hours, and then subsided again. The rows gradually disappeared. By seven days of culture, normal zone differences in cell density were lost, and epithelial cells began to migrate posteriorly beyond the equator. Although it was obvious that the regulation of cell proliferation was grossly disturbed in cultured lenses, we tested the action of IPR and camp derivatives in this system. The study was limited to the first 24 hours of incubation. Cell proliferation in lenses cultured in the presence of IPR and camp derivatives. The effects of different concentrations of IPR on mitotic activity after various intervals of culture are presented in Table II. Comparison is made between lenses of the same animal, one lens placed in control medium, and the other lens in medium containing IPR. The mitotic count in the experimental lens is expressed as per cent of the count in the control lens. Neither the test medium nor the control medium were changed during the culture period. Mitosis was inhibited after two hours of culture with IPR at concentrations be-

4 Volume 13 Number 3 Effects of IPR and cyclic AMP on cell division 213 Table II. Mitotic activity (per cent control ± S.E.) of rat lenses cultured in the presence of isoproterenol Drug concentration 1 x 10-4 M 5 x 10-5 M 2.5 x 10-5 M 1 x 10- r >M 5 x 10- (! M 1 x 10- r -M (15) 2 (12) (11) (12) hours 27 ± 4 26 ± 3 38 ± 4* Duration of incubation 4 hours (13) (12) (8) (9) The figures in parentheses represent the number of pairs of control and treated lenses in each group. "Difference from control value is significant at a probability level of p < (9) (15) (22) (18) hours 52 ± 76 ± 78 ± 85 ± (toxic) 5" tween 5 x 10~ a and 5 x 10~ (1 M; all phases were equally affected. When the incubation period was extended to four hours, mitotic activity was near normal. Twentyfour hours of exposure to the drug-containing medium resulted in only a marginal reduction of cell division without evidence of toxicity except at the highest concentration tested, 1 x 10" l M. At this level, IPR caused widespread cell death in the epithelial layer. The transient suppression of mitosis after two hours of incubation with IPR could be completely blocked by propranolol (Table III), demonstrating that the catecholamine-induced mitotic inhibition occurs through stimulation of beta-adrenergic receptors in the isolated lens. Propranolol alone did not affect mitotic counts in the lens epithelium. The inhibitory action of IPR was no longer evident at four hours of incubation, but if IPR was added after the lenses had been preincubated for two hours in control medium, mitosis was again suppressed at one and two hours after the addition of catecholamine (Table IV). The lens, therefore, remained sensitive to IPR between two and four hours of culture. It appeared that mitotic inhibition, once triggered, manifested itself rapidly but was transient in nature. Lenses cultured for 24 hours in IPRcontaining medium showed little effect of the drug except at the toxic concentration of 1 x 10~ 4 M. There was a small reduction of mitotic activity at drug concen- Table III. Propranolol blockade of IPR-induced mitotic suppression Mitotic activity Drug additions (per cent control ± S.E.) IPR (1 x 10-5 M) 28 ± 3* Propranolol (1 x 10" 5 M) (7) 94 ± 10 IPR + propranolol (11) 86+5 Lenses were placed initially in either control or propranolol-containing medium. After 30 minutes of incubation, IPR was added to the appropriate culture dishes and incubation was continued for two hours. The total duration of culture was 2% hours for all lenses in this experiment. The figures in parentheses represent the number of pairs of control and treated lenses in each group. "Difference from control value is significant at a probability level of p < Table IV. Response to IPR (5 x 10" 5 M) added after two hours of preincubation in control medium Treatment 2 hours preincubation + 1 houripr 2 hours preincubation + 2 hours IPR Mitotic activity (per cent control ± S.E.) * 54 ± 9«The figures in parentheses represent the number of pairs of control and treated lenses in each group. "Difference from control value is significant at a probability level of p < trations of 5 x 10- n and 2.5 x 10- r> M. To determine if this late mitotic depression was a consequence of inhibition of the DNA-synthesizing phase of the cell cycle, we examined the influence of IPR on H! -thymidine incorporation during the 24 hour culture period (Table V). No change in the number of DNA-synthesizing cells was detected at any interval; therefore, the reduction of mitotic activity seen at 24 hours was attributed to a cumulative non-

5 214 von Sallmann and Grimes Investigative Ophthalmology March MBcAMP I x I0' 3 M Prophase S Metaphase CD Telophase 100 o 200 a: DBcAMP Ix I0" 3 M Na Butyrate I x I0" 3 M j Jill ( TIME (hours) Fig. 2. Comparison of the influence of cyclic AMP derivatives and sodium butyrate on mitotic activity during 24 hours incubation. The left half of the figure illustrates the change in total mitotic counts. Effects on specific phases of mitoses are shown on the right. The vertical lines through each point or bar represent the standard error of the mean. j Table V. Effect of IPR (2.5 x 10" 5 M) on DNA synthesis and mitosis after varying intervals of culture Time in culture (hours) W-labeled cells (per cent control : S.E.) 99 ± 104 ± 101 ± 73 ± Mitoses (per cent control ± S.E.) (3) 78 ± 1* 105 ± 4 84 ± 9 66 ± 10" The figures in parentheses represent the number of pairs of control and treated lenses in each group. "Difference from control value is significant at a probability level of p < specific or toxic action of IPR or its degradation products. This interpretation received support from the observation that theophylline did not potentiate the late effect of IPR (Table VI). Theophylline alone caused a decrease of mitotic counts after 24 hours at a concentration of 1 x 10~ 3 M, but not at the lower level of 1 x 10-4 M. Lenses cultured in medium containing either DBcAMP or MBcAMP underwent an early transient suppression of mitotic activity which resembled that induced by IPR. Besides inhibiting the number of cells entering mitosis, the camp derivatives at high concentration directly affected the mitotic process. Fig. 2 compares the changes in total mitotic activity and the specific changes in prophase, metaphase, and telophase counts induced by DBcAMP, MBcAMP, and sodium butyrate at a concentration of 1 x 10" 3 M during 24 hours of incubation.

6 Volume 13 Number 3 Effects of IPR and cyclic AMP on cell division 215 Fig. 3, Butyrate-induced change in the appearance of cell nuclei. Epithelial cells after 24 hours of incubation in control medium (A) and in the presence of sodium butyrate (1 x lch3m) (B). (Feulgen stain, xl,600.) After two hours of culture, entry into mitosis was almost completely blocked by the camp derivatives; the number of prophases in the treated preparations fell to nearly zero. The inhibition was not revealed in the total mitotic counts because of the abnormal accumulation of metaphase and telophase figures, probably caused by a preceding or concurrent metaphase arrest. The shift in phase counts was of shorter duration than the mitotic inhibition; at three and four hours of incubation excess metaphase figures had disappeared and all phases of mitosis were equally reduced to approximately 25 per cent of control values. Mitotic inhibition, in turn, was no longer evident after six hours of incubation, and in the experiments with MBcAMP no further changes in mitotic activity were noticed after 16 and 24 hours. Lenses treated with DBcAMP, in contrast, showed significantly higher numbers of mitoses at the 16 and 24 hour intervals. This apparent stimulation was again associated with a pronounced increase of metaphase counts, whereas prophase and telophase Table VI. Mitotic activity of lenses cultured for 24 hours in the presence of IPR and theophylline Mitosis Drug addition (per cent control ± S.E., IPR (1 x 10-DM) (18) 85 ± 4 Theophylline (1 x 10-3M) (16) 73 ± 5 76 ± 4* IPR + theophylline (22) 78 ± 5 IPR (2.5x 10-BM) Theophylline (1 x 1(HM) (12) 104 ± 5 IPR + theophylline 81 ± 8 The figures in parentheses represent the number of pairs of control and treated lenses in each group. "Difference from control is significant at a probability level of p < counts did not differ significantly from control values. Sodium butyrate was tested because of its possible accumulation in the culture system as a product of the decomposition or enzymatic degradation of DBcAMP.fi"s It caused a similar late effect on the mitotic process. All phases of mitosis were suppressed slightly after incubation with sodium butyrate for two and four hours, but when the culture period was extended to 16 or 24 hours, metaphase counts rose to twice normal values. It is

7 216 von Sallmann and Grimes Investigative Ophthalmology March 1974 Table VII. Mitotic activity (per cent control ± S.E.) of lenses cultured in the presence of DBcAMP Duration (hours) hours preincubation + 4 hours DBcAMP 1 (5) (9) (10) (10) x 10-3 M 126 ± 19* 25 ± 4f 85 ± ± 14*, 155 ± 18*, t 4 ± 4f I x (9) (10) Drug concentration 10~*M I x KMM 54 ± 6f 62 ± 3t 79 ± ± 6 94 ± 4 72 ± 7f 97 ± 9 The figures in parentheses represent the number of pairs of control and treated lenses in each group. "Abnormal accumulation of metaphase figures. tdifference from control values is significant at a probability level of p < I x 10-"M 98 ± ± 7 possible, therefore, that the interference with mitotic division seen after prolonged incubation with DBcAMP resulted from contamination with butyrate. Lenses treated with 1 x 10~ 3 M sodium butyrate demonstrated a characteristic morphologic alteration of all cell nuclei in the epithelial preprations at each incubation period examined. Small clumps of densely stained material were scattered throughout the nucleoplasm (Fig. 3). Such change was not seen in lenses incubated with 1 x 10~ 4 M sodium butyrate (four hours), nor in cells exposed to the butyryl derivatives of camp. Lower concentrations of DBcAMP (1 x 10~ 4 and 1 x ICH'M) produced less marked but statistically significant mitotic inhibition at two and four hours without signs of metaphase accumulation (Table VII). The lower levels of DBcAMP did not alter mitotic activity at 16 and 24 hours. The effect of DBcAMP (1 x 10 3 M) on DNA synthesis was also tested at 4, 8, 16, and 24 hours. The number of cells incorporating H 3 -thymidine in treated lenses did not differ from control lenses at any of the four intervals (data not shown). The mitotic inhibition induced by the cyclic AMP derivatives was more protracted than that encountered with IPR, but did not last longer than four to six hours. When DBcAMP was added to the medium after lenses had been incubated for 20 hours in control medium, mitotic activity fell to 4 per cent of control values four hours later (Table VII). The lens epithelium, therefore, does not lose its ability to respond to fresh addition of DBcAMP during a prolonged period of culture. In addition to their action on cell division, both MBcAMP and DBcAMP prevented the destruction of cells in the meridional rows. Lenses incubated for 24 hours with these compounds at a concentration of 1 x 10~ ;i M showed some loss of the ordered arrangement of the row cells, but there was no evidence of nuclear disintegration at this time nor after shorter periods of incubation. Disintegrated nuclei were present without exception in the meridional rows of all control and other experimentally treated lenses incubated for longer than six hours. Selective destruction of the meridional rows under conditions of culture and their preservation in the presence of camp derivatives suggests that these differentiating cells have certain distinctive metabolic requirements. Discussion Studies of the isolated rat lens in culture were undertaken to provide evidence for some form of hormonal regulation of cell proliferation in the lens epithelium. Previous experiments in the living rat had implied the existence of such a mechanism mediated by cyclic AMP. As mentioned before, Makman and Kern 3 identified catecholamine-stimulated adenylate cyclase

8 Volume 13 Number 3 Effects of IPR and cyclic AMP on cell division 217 of high specific activity in the combined capsule-epithelium portion of the rat lens. The data presented here demonstrate the presence of elements of a regulatory mechanism in the lens itself. IPR, in vitro, transiently inhibited the passage of cells from the G 2 phase of the cell cycle to mitosis, and propranolol blocked this response. There are, therefore, beta-adrenergic receptors in lens cells, the stimulation of which influence mitotic activity. Furthermore, since the catecholamine-induced mitotic inhibition was mimicked by camp derivatives, participation of camp in the process is supported. In vivo, the IPR-induced suppression of cell division was initiated in the Gi phase of the cycle with reduction of the number of DNA-synthesizing cells at 16 hours after administration of the drug: the fall in mitotic counts was detected eight hours later. 1 In cultured lenses, we did not discover any change in number of cells entering the S phase during 24 hours of incubation with IPR or DBcAMP. The small decrease in mitoses seen at the 24 hour interval in vitro with higher concentrations of IPR was interpreted as a nonspecific toxic effect of the drug. No explanation can be offered, as yet, for the failure to detect a Gi inhibition in vitro, but it may be related to the marked disturbance of the normal proliferative process brought about by the conditions of culture. The transient G 2 block induced by IPR in the cultured lens was not seen when the catecholamine was given to the living animal. It is possible that an early shortlasting mitotic inhibition passed undetected in vivo because, in those experiments, lenses were not examined until four hours after IPR was given. In rat salivary gland, a tissue which uniquely responds to IPR in vivo with a massive stimulation of DNA synthesis and mitosis, a transient G. block, and metaphase arrest occurs within two hours after catecholamine injection. 9 Inhibition of mitotic division in the G 2 period has been studied extensively in skin and attributed to the action of epidermal chalones. According to Voorhees and coworkers IPR, through stimulation of beta receptors, can mimic the action of chalones in producing a G 2 block in cultured epidermis. In their experiments DBcAMP also produced the same response. Leeson and Voaden 13 in studies of the chalone mechanism in the cultured rabbit lens, reported that catecholamines alone induced a transient G 2 mitotic inhibition which appears to be similar in extent and duration to the response we observed in the rat lens. Epinephrine was approximately lox more effective than norepinephrine in evoking the response. They noted that before the onset of inhibition, within 10 to 15 minutes of the addition of high doses of epinephrine, metaphase and telophase figures increased abnormally. In our experiments, prolongation of the mitotic process was not seen after one hour of incubation with IPR, the earliest interval examined, but was pronounced in the presence of camp derivatives. These agents had consistently longer lasting effects than did IPR. Rapid manifestation of the G 2 block together with signs of interference with chromosome movement suggest that elevation of the camp level influences the assembly or stability of the mitotic apparatus, possibly through the activation of protein kinases. Cyclic AMPinduced shifts in the intracellular concentrations of calcium ion may also play a determining role. 14 In cultured thymocytes a complex interaction between calcium concentration and camp levels has been reported to govern the progression of cells from the G 2 period to mitosis. 15 Recent reports indicate that DBcAMP is converted to N n -monobutyryl camp by spontaneous 6 or enzymatic 7-8 deacylation and that the monobutyryl compound is the active derivative within the cell. We found MBcAMP to be equally as effective as DBcAMP in inducing mitotic suppression in the lens. Moreover, after extended incubation, DBcAMP at high concentration caused an abnormal prolongation of metaphase. Since butyrate alone produced a

9 218 von Sallmann and Grimes Investigative Ophthalmology March 1974 similar late accumulation of metaphases, this effect of DBcAMP may be due to increasing levels of butyrate arising from the degradation of the dibutyryl compound. Transient inhibition of the cell cycle in the Go phase is of doubtful significance as a physiologic population control mechanism. The presence of this response, however, does serve to demonstrate that stimulation with beta-adrenergic agonists and elevation of camp levels can directly affect the cell cycle of the lens epithelium. Further investigation is necessary to determine if a more meaningful hormonal regulation exists. REFERENCES 1. von Sallmann, L., and Grimes, P.: Isoproterenol-induced changes of cell proliferation in rat lens epithelium, INVEST. OPHTHALMOL. 10: 943, Grimes, P., and von Sallmann, L.: Possible cyclic adenosine monophosphate mediation in isbproterenol-induced suppression of cell division in rat lens epithelium, INVEST. OPHTHAL- MOL. 11: 231, Makman, M. H., and Kern, H. L.: Adenyl cyclase of lens, cultured lens epithelial cells, and retina; stimulation by catecholamines, Fed. Proc. 30: 1205 (Part II), (Abstr.) 4: Merola, L. O., Kern, H. L., and Kinoshita, J.: The effect of calcium on the cations of the calf lens, Arch. Ophthalmol. 63: 830, Thoft, R. A., and Kinoshita, J. H.: The extracellular space of the lens, Exp. Eye Res. 4: 287, Swislocki, N.: Decomposition of dibutyryl cyclic AMP in aqueous buffers, Anal. Biochem. 38: 260, Kaukel, E., Mundhenk, K., and Hilz, H.: N u monobutyryladenosine 3':5'-monophosphate as the biologically active derivative of dibutyryladenosine 3':5'-monophosphate in HeLa S3 cells, Eur. J. Biochem. 27: 197, Blecher, M., and Hunt, N. H.: Enzymatic deacylation of mono- and dibutyryl derivatives of cyclic adenosine 3':5'-monophosphate by extracts of rat tissues, J. Biol. Chem. 247: 7479, Radley, J. M., and Hodgson, G. S.: Effect of isoprenaline on cells in different phases of the mitotic cycle, Exp. Cell Res. 69: 148, Voorhees, T. T., Duell, E. A., Bass, L. T., et al.: Inhibition of epidermal cell division by isoproterenol, dibutyryl cyclic AMP, and theophylline, Clin. Res. 19: 682, (Abstr.) 11. Powell, J. A., Duell, E. A., and Voorhees, J. J.: Beta-adrenergic stimulation of endogenous epidermal cyclic AMP formation, Arch. Dermatol. 104: 359, Voorhees, J. J., Duell, E. A., and Kelsey, W. H.: Dibutyryl cyclic AMP inhibition of epidermal cell division, Arch Dermatol. 105: 384, Leeson, S. J., and Voaden, M. J.: A chalone in the mammalian lens. II. Relative effects of adrenaline and noradrenaline on cell division in the rabbit lens, Exp. Eye Res. 9: 67, Rasmussen, H., Kurokawa, K., Mason, J., et al.: Cyclic AMP, calcium, and cell activation, In: Calcium, Parathyroid Hormone and the Calcitonins, Talmage, R., and Munson, P., editors. Amsterdam, 1972, Excerpta Medica Foundation, p Whitfield, J. F., MacManus, J. P., Braceland, B., et al.: The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E,, J. Cell Physiol. 79: 353, 1972.