- Email: [email protected]
Intracellular Levels of Adenosine 3′:5′-Cyclic Monophosphate in the Dorsal Iris of the Adult Newt, During Lens Regeneration
Differentia tion
Differentiation 17, 117-120 (1980)
0 Springer-Verlag 1980
Intracellular Levels of Adenosine 3’:5’-Cyclic Monophosphate in the Dorsal Iris of the Adult Newt, During Lens Regeneration F. M. VELAZQUEZ and J. R. ORTiZ’ Department of Biology, University of Puerto Rico, N o Piedras, Puerto Rico 00931
The intracellular levels of adenosine 3’5’-cyclic monophosphate (CAMP)were measured in the dorsal iris of the adult newt, during the first 20 days of lens regeneration. It was found that by day 2 after lens removal there is a significant drop in the levels of CAMP.After day 2 the levels of the nucleotide increase and by day 3 they are higher than those detected on day 0. The levels of CAMP remain high up to day 8. From day 8 to day 9 there is a second drop. From day 9 to day 20 the levels of cAMP did not differ significantly from the value obtained for day 0, except for days 10, 12, and 15. The period of high levels of cAMP coincides with the period of depigmentation of iris epithelial cells, the key event of lens regeneration.
Introduction After lentectomy of the adult newt eye, the melanocytes of the pupillary margin of the dorsal iris undergo dedifferentiation and later redifferentiate into lens cells [l-31. It is known that during the dedifferentiation phase those cells discharge melanin granules, fragments of cytoplasm surrounded by membranes and other cytoplasmic components [5,6]. During the dedifferentiation phase, an oriented network of microfilaments appears in the cortical layers of the iris epithelial cell cytoplasm and a population of microtubules can be found in the cell projections [4]. It has been suggested that the microtubular microfibrillar system may play an important role in the morphologic alterations of iris epithelial cells during the dedifferentiation phase [4]. The morphologic features of dedifferentiating iris epithelial cells which occur in vivo during lens regeneration can be mimicked in cultured iris epithelial cells by administering a number of agents known to increase the intracellular levels of adenosine 3’5’-cyclic monophosphate (CAMP) (7-91. Furthermore, there is some evidence for the involvement of microtubules and microfilaments in the regu1
Author to whom reprint request should be addressed
lation of the cell shape by cAMP [lo]. Based on the results described above we decided to measure the intracellular levels of cAMP in the dorsal iris in vivo during the first 20 days of the regeneration process. There has been a report of similar measurements for the first five days after lentectomy [ll]. Methods Adult newts, Notophthalmus viridescens were obtained from Bill Lee’s Newt farm, Oak Ridge, Tennessee. They were kept in 10-gallon aquaria at 21 f 1”C and were fed with beef liver. Before the surgery the animals were anesthetized with Tricaine (ethyl-m-aminobenzoate methane sulfonate, Sigma Chemical Co.) at a concentration of 0.4 g/liter. Lentectomy and separation of the dorsal iris was done as described previously [12]. Immediately after removal, the dorsal iris was placed in cold (4” C) 80% phosphate buffer saline (PBS) (Grand Island Biological Co.). Measurements of the intracellular levels of cAMP and the isolation of the binding protein were done according to the procedures described by Gilman [13]. The same protein kinase or binding protein was used in all determinations and its activity or binding capacity was measured every time it was used. The average counts per minute (binding capacity) for 20 p1 of the enzyme when reacted with 25 111 of radioactive CAMPcontaining 1.33 X 104 pmol of the nucleotide (specific activity 37.7 Cilmmol of CAMP, New England Nuclear Co.) were 16,392 cpdsample with a standard deviation of k 150. As a protein kinase inhibitor, a solution of albumin (15 mg/ml) was used [14]. Total protein concentration was measured for each
0301-4681/80/0017/0117/$01.OO
G1 phase and then further proceed in cell cycles [3,5, 111. According to our data this transition appears to occur around the time of the minimum level of CAMP. This observation poses the question as to whether the
r(
I
I
I
I
F. M. Veliizquez and J. R. Ortiz: Intracellular Levels of cAMP During Lens Regeneration
systems at different cellular levels [21-251. However, since a relatively high level of CAMP is associated with the normal state of the iris, when the synthesisof RNA and protein is minimum [ l l , 16,201, one must admit that no unequivocalcorrelation existsbetween the level of cAMP and the extent of RNA and protein synthesis in this system. Either a number of other factors are involved in the regulation of the correlation is fortuitous. The interpretation of data presented here is somewhat different from that of Thorpe et al. [ll] who suggested a negative control of the enhancement of RNA synthesis by CAMP. The proliferative activityor DNAreplication in this system is generally associated with the period of relatively high levels of CAMP, except in the normal iris where the minimum proliferative activity is accompanied by a relatively high level of CAMP.It is impressive that the onset of DNA replication around days 3 to 4 in the iris epithelium [11,26-29] is preceded by a rapid increase of the cAMPlevel, as pointed out by Thorpeet al. [ll]. This aspect of the present system is in agreement with many studies which demonstrate a correlation between the pattern of DNA replication or proliferative activity and the cAMP level [30-351. It is, however, unlikely that the observed decrease of the cAMP level on day 9 is correlated with a decrease in proliferation because in this system the period of active proliferation is known to last at least until day 15 [28, 361, Probably the most pertinent observation made in the present study is that the highest levels of CAMP are found between days 3 and 8. Only during this period does the value remain continuously and significantly above the normal value. Histologicstudies suggest that most of the depigmentation of iris epithelial cells, the key event of dedifferentiation in this system, is completed by days 8 to 10 [3]. Thus our results support the notion that cAMPisinvolvedindedifferentiationofiris epithelial cells by possibly affecting their microtubular-microfibrillar system [7-lo]. It is well known that CAMP,microtubules, and microfilamentsare involved in cellular morphogenesis in several other biologic systems [37-421. Acknowledgements. This research was sponsored by NIH Grant RR-8102 and the Office of Coordination of Graduate Studies and Research, University of Puerto Rim, KOPiedras Campus. The author’sthanksare due toDr. Rudolf Achazi, ZoologicalInstitute of University of Miinster, for instruction of the method, and Mr. Francisco Dueno for participating in the initial stages of this work when he was a trainee of the MBS Program. We would also like to thank Dr. Isaura M e n , Departamento de Biologfa Celular, CIEA del IPN, Mexico, and Dr. Tune0 Yamada, Swiss Institute for Experimental Cancer Research, Epalinges, for criticallyreading the manuscript.
119
References 1. ReyerRW (1954) Regenerationofthelensinthe amphibianeye. Quart Rev Biol29 : 1 2. YamadaT (1967)Cellular and subcellulareventsin Wolffian lens regeneration. Current Topics Develop Biol2: 247 3. YamadaT (1977) Control mechanismsin cell-typeconversionin newt lens regeneration. Monographs Develop Bioll3: 1 4. DumontJN,YamadaT(1972)Dedifferentiationofirisepithelial cells. Develop Biol29: 385 5. YamadaT(1972) Controlmechanismsincellularmetaplasia.In: Harris R, AUinP, Viza 0 (eds) Proceedingsof 1stConferenceof Cell Differentiation, Munksugaard, Copenhagen, p 56 6. Yamada T (1976) Dedifferentiation associated with cell-type conversion in the newt lens regenerating system. Muller-BCrat Progress in the differentiation research. North-Holland, Amsterdam, p 355 7. Ortiz JR (1973) Dedifferentiationof iris epithelial cells in vitro, Ph.D. Dissertation. University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences 8. Ortiz JR, Yamada T, Hsie AW (1973) Induction of the stellate configuration in cultured iris epithelial cells by adenosine and compounds related to adenosine 3’:S-cyclic monophosphate. Proc Natl Acad Sci USA 70 :2286 9. Ortiz JR. Yamada T (1975) Synergisticeffect of adenosine and compoundsrelated to adenosine 3’:5’-cyclic monophosphate of dedifferentiating iris epithelial cells in culture. Differentiation 4: 135 10. Ortiz JR; Connelly GT (1977) The effect of colchicine on the inductionofthestellateconfigurationindorsalirisepithelial~~s of the newt Notophthulmusvirisdescens,invitro. Differentiation 9: 175 11. Thorpe CW, Bond JS, Collins JM (1974) Early events in lens regeneration. Changes in cyclic AMP concentrations during initiation of RNA and DNA synthesis. Biochim Biophys Acta 340: 413 12. Reese DH, Puccia E, YamadaT (1969) Activation of ribosomal RNA synthesis in the initiation of Wolffian lens regeneration. J Exptl Zoo1 170: 259 13. Gilman AC (1970) A protein binding assay for adenosine 3’5‘-cyclicmonophosphate. Proc Natl Acad Sci USA 67: 305 14. RodriguezN, Renaud F(1980) On the possible role of serotonin in the regulation of cilia regeneration. J Cell Biol85 : 242 15. Miller GL (1959) Protein determination for a large number of samples. Anal Chem 31 : 964 16. Reese DH (1973) In vitro initiationin the newt iris of some early molecular events of lens regeneration. Exptl Eye Res 17: 435 17. Collins JM (1972) Amplification of ribosomal ribonucleic acid cistrons in the regeneration of the lens of Triturus.Biochemistry 11: 1259 18. Dumont JN, Yamada T, Cone V (1970) Alteration of the nucleolar ultrastructure in iris epithelial cells duringinitiationof Wolffian lens regeneration. J Exptl Zoo1 174: 184 19. Yamada T, Takata C (1973) An autoradiographic study of protein synthesisin regenerativetissuetransformationof irisinto lens in the newt. Develop Biol8 : 358 20. YamadaT, KarasakiS (1963)Nuclear RNAsynthesisinnewt iris cells engaged in regenerative transformation into lens cells. Develop Biol7 :595 21. LanganTA(1969)Phosphorylationof liverhistonefollowing the administrationof glucagonand insulin. Proc Natl Acad Sci USA 64:1272 22. Foret JE, Babich GL (1973) Effect of dibutyryl cyclic AMP and related compounds on newt limb regeneration blastemas in vitro. Oncology 28: 89
120
F. M. Velazquez and J. R. Ortiz: Intracellular Levels of CAMP During Lens Regeneration
23. RosenfeldMG, AbrassIB,Mendelsohn J,RoosBA.BooneRR, Garren LD (1972) Control of transcription of RNA rich in polyadenylic acid in human lymphocytes. Proc Natl Acad Sci USA 69 : 2306 24. Dokas LA, Kleinsmith U (1971) Adenosine 3':5'-monophosphate increases capacity for RNA synthesis in rat liver nuclei. Science 172 : 1237 25. MoralesMH, BrownPC, PapaconstantinouJ (1978) Regulation of a-fetoprotein synthesis in cultured mouse hepatoma cells (Hepa-2). J Cell Biol79: 352a 26. EisenbergS, YamadaT(1966)AstudyofDNAsynthesisduring the transformationof the irisinto lens in the lentectomizednewt. J Exptl Zoo1 162: 353 27. Yamada T, Roesel ME (1%9) Activation of DNA replication in the iris epithelium by lens removal. J Exptl Zoo1 171: 425 28. Reyer RW (1971) DNA synthesis and the incorporation of labeled iris cells into the lens during lens regeneration in adult newts. Develop Biol24: 535 29. Eguchi C. Shingai R (1971) Cellular analysis on localization of lens-formingpotency in newt iris epithelium. Develop Growth Differentiation 13: 337 30. Whitfield JF, MacManus JP, Youdale T. Franks DF (1970) The role of calcium and cyclic AMP in the stimulatory action of parathyroid hormone on thymic lymphocytes. J Cell Physiol 77: 103 31. MacManus JP, Whitfield JF, Youdale T (1970) Stimulation by epinephrine of adenylcyclase activity, cyclic AMP formation, DNA synthesis and cell proliferationin population of rat thymic lymphocytes. J Cell Physiol77 : 103 32. Foret TE (1973) Stimulationand retardation of limb regeneration by adenosine 3':5'-cyclic monophosphate and related compounds. Oncology 27 : 153 33. Naimey SN (1978) The effects of the levels of adenosine 3'5'-cyclic monophosphate and related compounds. M.S. Dissertation, Department of Biology, University of Puerto Rico
34. Taban C, Cathieni M, Schorderet M (1978) Cyclic AMP and noradrenaline sensitivity fluctuations in regenerating tissue. Nature 271 : 470 35. Creighton MC, Trevithiek JR (1974) Effect of cyclic AMP, caffeine and theophylline on differentiation of lens epithelial cells. Nature 249: 767 36. Yamada T, Roesel ME (1971) Control of mitotoc activity in Wolffian lens regeneration. J Exptl Zoo1 117: 119 37. Hsie AW, Jones C, Puck TT (1968) Further changes in differentiation state accompanying the conversion of Chinese hamster cells to fibroblasticform by dibutyryl adenosine cyclic 3'5'-monophosphate and hormones. Proc Natl Acad Sci USA 68: 1648 38. Puck lT,Waldren C, Hsie AW (1972) Membrane dynamics in the action of dibutyryl adenosine 3':5'-cyclic monophosphate and testosterone on mammalian cells. Proc Natl Acad Sci USA 69: 1943 39. Prasad KN, Hsie AW (1971) Morphologic differentiation of mouse neuroblastoma cells induced in vitro by dibutyryl adenosine 3'5'-cyclic monophosphate. Nature 233 : 141 40. Puck=, JonesC( 1974)CyclicAMPandmicrotubulardynamics in cultured mammalian cells. In: Braun W, Lichtenstein LM. Parker CW (eds) Cyclic AMP, cell growth and the immune response. Springer, Berlin Heidelberg New York, p 338 41. YokotaT, Gots JS (1970) Requirement of adenosine3':5'-cyclic monophosphate for flagella formation in Escherichiu coli and Salmonella typhimuriurn. J BacteriollO3 :513 42. Wessels NK, Spooner BS, Ash JF, Bradley MO, Luduena MA, Taylor EL, Wrenn JR, Yamada KM (1971) Microfilaments in cellular and developmental processes. Science 171: 135
Received October 1979/Accepted May 1980
Differentiation 17, 117-120 (1980)
0 Springer-Verlag 1980
Intracellular Levels of Adenosine 3’:5’-Cyclic Monophosphate in the Dorsal Iris of the Adult Newt, During Lens Regeneration F. M. VELAZQUEZ and J. R. ORTiZ’ Department of Biology, University of Puerto Rico, N o Piedras, Puerto Rico 00931
The intracellular levels of adenosine 3’5’-cyclic monophosphate (CAMP)were measured in the dorsal iris of the adult newt, during the first 20 days of lens regeneration. It was found that by day 2 after lens removal there is a significant drop in the levels of CAMP.After day 2 the levels of the nucleotide increase and by day 3 they are higher than those detected on day 0. The levels of CAMP remain high up to day 8. From day 8 to day 9 there is a second drop. From day 9 to day 20 the levels of cAMP did not differ significantly from the value obtained for day 0, except for days 10, 12, and 15. The period of high levels of cAMP coincides with the period of depigmentation of iris epithelial cells, the key event of lens regeneration.
Introduction After lentectomy of the adult newt eye, the melanocytes of the pupillary margin of the dorsal iris undergo dedifferentiation and later redifferentiate into lens cells [l-31. It is known that during the dedifferentiation phase those cells discharge melanin granules, fragments of cytoplasm surrounded by membranes and other cytoplasmic components [5,6]. During the dedifferentiation phase, an oriented network of microfilaments appears in the cortical layers of the iris epithelial cell cytoplasm and a population of microtubules can be found in the cell projections [4]. It has been suggested that the microtubular microfibrillar system may play an important role in the morphologic alterations of iris epithelial cells during the dedifferentiation phase [4]. The morphologic features of dedifferentiating iris epithelial cells which occur in vivo during lens regeneration can be mimicked in cultured iris epithelial cells by administering a number of agents known to increase the intracellular levels of adenosine 3’5’-cyclic monophosphate (CAMP) (7-91. Furthermore, there is some evidence for the involvement of microtubules and microfilaments in the regu1
Author to whom reprint request should be addressed
lation of the cell shape by cAMP [lo]. Based on the results described above we decided to measure the intracellular levels of cAMP in the dorsal iris in vivo during the first 20 days of the regeneration process. There has been a report of similar measurements for the first five days after lentectomy [ll]. Methods Adult newts, Notophthalmus viridescens were obtained from Bill Lee’s Newt farm, Oak Ridge, Tennessee. They were kept in 10-gallon aquaria at 21 f 1”C and were fed with beef liver. Before the surgery the animals were anesthetized with Tricaine (ethyl-m-aminobenzoate methane sulfonate, Sigma Chemical Co.) at a concentration of 0.4 g/liter. Lentectomy and separation of the dorsal iris was done as described previously [12]. Immediately after removal, the dorsal iris was placed in cold (4” C) 80% phosphate buffer saline (PBS) (Grand Island Biological Co.). Measurements of the intracellular levels of cAMP and the isolation of the binding protein were done according to the procedures described by Gilman [13]. The same protein kinase or binding protein was used in all determinations and its activity or binding capacity was measured every time it was used. The average counts per minute (binding capacity) for 20 p1 of the enzyme when reacted with 25 111 of radioactive CAMPcontaining 1.33 X 104 pmol of the nucleotide (specific activity 37.7 Cilmmol of CAMP, New England Nuclear Co.) were 16,392 cpdsample with a standard deviation of k 150. As a protein kinase inhibitor, a solution of albumin (15 mg/ml) was used [14]. Total protein concentration was measured for each
0301-4681/80/0017/0117/$01.OO
G1 phase and then further proceed in cell cycles [3,5, 111. According to our data this transition appears to occur around the time of the minimum level of CAMP. This observation poses the question as to whether the
r(
I
I
I
I
F. M. Veliizquez and J. R. Ortiz: Intracellular Levels of cAMP During Lens Regeneration
systems at different cellular levels [21-251. However, since a relatively high level of CAMP is associated with the normal state of the iris, when the synthesisof RNA and protein is minimum [ l l , 16,201, one must admit that no unequivocalcorrelation existsbetween the level of cAMP and the extent of RNA and protein synthesis in this system. Either a number of other factors are involved in the regulation of the correlation is fortuitous. The interpretation of data presented here is somewhat different from that of Thorpe et al. [ll] who suggested a negative control of the enhancement of RNA synthesis by CAMP. The proliferative activityor DNAreplication in this system is generally associated with the period of relatively high levels of CAMP, except in the normal iris where the minimum proliferative activity is accompanied by a relatively high level of CAMP.It is impressive that the onset of DNA replication around days 3 to 4 in the iris epithelium [11,26-29] is preceded by a rapid increase of the cAMPlevel, as pointed out by Thorpeet al. [ll]. This aspect of the present system is in agreement with many studies which demonstrate a correlation between the pattern of DNA replication or proliferative activity and the cAMP level [30-351. It is, however, unlikely that the observed decrease of the cAMP level on day 9 is correlated with a decrease in proliferation because in this system the period of active proliferation is known to last at least until day 15 [28, 361, Probably the most pertinent observation made in the present study is that the highest levels of CAMP are found between days 3 and 8. Only during this period does the value remain continuously and significantly above the normal value. Histologicstudies suggest that most of the depigmentation of iris epithelial cells, the key event of dedifferentiation in this system, is completed by days 8 to 10 [3]. Thus our results support the notion that cAMPisinvolvedindedifferentiationofiris epithelial cells by possibly affecting their microtubular-microfibrillar system [7-lo]. It is well known that CAMP,microtubules, and microfilamentsare involved in cellular morphogenesis in several other biologic systems [37-421. Acknowledgements. This research was sponsored by NIH Grant RR-8102 and the Office of Coordination of Graduate Studies and Research, University of Puerto Rim, KOPiedras Campus. The author’sthanksare due toDr. Rudolf Achazi, ZoologicalInstitute of University of Miinster, for instruction of the method, and Mr. Francisco Dueno for participating in the initial stages of this work when he was a trainee of the MBS Program. We would also like to thank Dr. Isaura M e n , Departamento de Biologfa Celular, CIEA del IPN, Mexico, and Dr. Tune0 Yamada, Swiss Institute for Experimental Cancer Research, Epalinges, for criticallyreading the manuscript.
119
References 1. ReyerRW (1954) Regenerationofthelensinthe amphibianeye. Quart Rev Biol29 : 1 2. YamadaT (1967)Cellular and subcellulareventsin Wolffian lens regeneration. Current Topics Develop Biol2: 247 3. YamadaT (1977) Control mechanismsin cell-typeconversionin newt lens regeneration. Monographs Develop Bioll3: 1 4. DumontJN,YamadaT(1972)Dedifferentiationofirisepithelial cells. Develop Biol29: 385 5. YamadaT(1972) Controlmechanismsincellularmetaplasia.In: Harris R, AUinP, Viza 0 (eds) Proceedingsof 1stConferenceof Cell Differentiation, Munksugaard, Copenhagen, p 56 6. Yamada T (1976) Dedifferentiation associated with cell-type conversion in the newt lens regenerating system. Muller-BCrat Progress in the differentiation research. North-Holland, Amsterdam, p 355 7. Ortiz JR (1973) Dedifferentiationof iris epithelial cells in vitro, Ph.D. Dissertation. University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences 8. Ortiz JR, Yamada T, Hsie AW (1973) Induction of the stellate configuration in cultured iris epithelial cells by adenosine and compounds related to adenosine 3’:S-cyclic monophosphate. Proc Natl Acad Sci USA 70 :2286 9. Ortiz JR. Yamada T (1975) Synergisticeffect of adenosine and compoundsrelated to adenosine 3’:5’-cyclic monophosphate of dedifferentiating iris epithelial cells in culture. Differentiation 4: 135 10. Ortiz JR; Connelly GT (1977) The effect of colchicine on the inductionofthestellateconfigurationindorsalirisepithelial~~s of the newt Notophthulmusvirisdescens,invitro. Differentiation 9: 175 11. Thorpe CW, Bond JS, Collins JM (1974) Early events in lens regeneration. Changes in cyclic AMP concentrations during initiation of RNA and DNA synthesis. Biochim Biophys Acta 340: 413 12. Reese DH, Puccia E, YamadaT (1969) Activation of ribosomal RNA synthesis in the initiation of Wolffian lens regeneration. J Exptl Zoo1 170: 259 13. Gilman AC (1970) A protein binding assay for adenosine 3’5‘-cyclicmonophosphate. Proc Natl Acad Sci USA 67: 305 14. RodriguezN, Renaud F(1980) On the possible role of serotonin in the regulation of cilia regeneration. J Cell Biol85 : 242 15. Miller GL (1959) Protein determination for a large number of samples. Anal Chem 31 : 964 16. Reese DH (1973) In vitro initiationin the newt iris of some early molecular events of lens regeneration. Exptl Eye Res 17: 435 17. Collins JM (1972) Amplification of ribosomal ribonucleic acid cistrons in the regeneration of the lens of Triturus.Biochemistry 11: 1259 18. Dumont JN, Yamada T, Cone V (1970) Alteration of the nucleolar ultrastructure in iris epithelial cells duringinitiationof Wolffian lens regeneration. J Exptl Zoo1 174: 184 19. Yamada T, Takata C (1973) An autoradiographic study of protein synthesisin regenerativetissuetransformationof irisinto lens in the newt. Develop Biol8 : 358 20. YamadaT, KarasakiS (1963)Nuclear RNAsynthesisinnewt iris cells engaged in regenerative transformation into lens cells. Develop Biol7 :595 21. LanganTA(1969)Phosphorylationof liverhistonefollowing the administrationof glucagonand insulin. Proc Natl Acad Sci USA 64:1272 22. Foret JE, Babich GL (1973) Effect of dibutyryl cyclic AMP and related compounds on newt limb regeneration blastemas in vitro. Oncology 28: 89
120
F. M. Velazquez and J. R. Ortiz: Intracellular Levels of CAMP During Lens Regeneration
23. RosenfeldMG, AbrassIB,Mendelsohn J,RoosBA.BooneRR, Garren LD (1972) Control of transcription of RNA rich in polyadenylic acid in human lymphocytes. Proc Natl Acad Sci USA 69 : 2306 24. Dokas LA, Kleinsmith U (1971) Adenosine 3':5'-monophosphate increases capacity for RNA synthesis in rat liver nuclei. Science 172 : 1237 25. MoralesMH, BrownPC, PapaconstantinouJ (1978) Regulation of a-fetoprotein synthesis in cultured mouse hepatoma cells (Hepa-2). J Cell Biol79: 352a 26. EisenbergS, YamadaT(1966)AstudyofDNAsynthesisduring the transformationof the irisinto lens in the lentectomizednewt. J Exptl Zoo1 162: 353 27. Yamada T, Roesel ME (1%9) Activation of DNA replication in the iris epithelium by lens removal. J Exptl Zoo1 171: 425 28. Reyer RW (1971) DNA synthesis and the incorporation of labeled iris cells into the lens during lens regeneration in adult newts. Develop Biol24: 535 29. Eguchi C. Shingai R (1971) Cellular analysis on localization of lens-formingpotency in newt iris epithelium. Develop Growth Differentiation 13: 337 30. Whitfield JF, MacManus JP, Youdale T. Franks DF (1970) The role of calcium and cyclic AMP in the stimulatory action of parathyroid hormone on thymic lymphocytes. J Cell Physiol 77: 103 31. MacManus JP, Whitfield JF, Youdale T (1970) Stimulation by epinephrine of adenylcyclase activity, cyclic AMP formation, DNA synthesis and cell proliferationin population of rat thymic lymphocytes. J Cell Physiol77 : 103 32. Foret TE (1973) Stimulationand retardation of limb regeneration by adenosine 3':5'-cyclic monophosphate and related compounds. Oncology 27 : 153 33. Naimey SN (1978) The effects of the levels of adenosine 3'5'-cyclic monophosphate and related compounds. M.S. Dissertation, Department of Biology, University of Puerto Rico
34. Taban C, Cathieni M, Schorderet M (1978) Cyclic AMP and noradrenaline sensitivity fluctuations in regenerating tissue. Nature 271 : 470 35. Creighton MC, Trevithiek JR (1974) Effect of cyclic AMP, caffeine and theophylline on differentiation of lens epithelial cells. Nature 249: 767 36. Yamada T, Roesel ME (1971) Control of mitotoc activity in Wolffian lens regeneration. J Exptl Zoo1 117: 119 37. Hsie AW, Jones C, Puck TT (1968) Further changes in differentiation state accompanying the conversion of Chinese hamster cells to fibroblasticform by dibutyryl adenosine cyclic 3'5'-monophosphate and hormones. Proc Natl Acad Sci USA 68: 1648 38. Puck lT,Waldren C, Hsie AW (1972) Membrane dynamics in the action of dibutyryl adenosine 3':5'-cyclic monophosphate and testosterone on mammalian cells. Proc Natl Acad Sci USA 69: 1943 39. Prasad KN, Hsie AW (1971) Morphologic differentiation of mouse neuroblastoma cells induced in vitro by dibutyryl adenosine 3'5'-cyclic monophosphate. Nature 233 : 141 40. Puck=, JonesC( 1974)CyclicAMPandmicrotubulardynamics in cultured mammalian cells. In: Braun W, Lichtenstein LM. Parker CW (eds) Cyclic AMP, cell growth and the immune response. Springer, Berlin Heidelberg New York, p 338 41. YokotaT, Gots JS (1970) Requirement of adenosine3':5'-cyclic monophosphate for flagella formation in Escherichiu coli and Salmonella typhimuriurn. J BacteriollO3 :513 42. Wessels NK, Spooner BS, Ash JF, Bradley MO, Luduena MA, Taylor EL, Wrenn JR, Yamada KM (1971) Microfilaments in cellular and developmental processes. Science 171: 135
Received October 1979/Accepted May 1980