Continuous-Perifusion tissue culture of fetal and adult pancreas of the lizard Anolis carolinensis.код для вставкиСкачать
THE ANATOMICAL RECORD 203:165-173 (1982) Continuous-PerifusionTissue Culture of Fetal and Adult Pancreas of the Lizard Anolis carolinensis WILLIAM R. RHOTEN Department of Anatomy, CMDNJ-New Jersey Medical School, Newark, NJ 07103 ABSTRACT The differentiation of the fetal saurian pancreas in continuousperifusion tissue culture (CPTC)was examined. Splenic pancreases from 24-day postoviposition fetuses of the green anole,Anolis carolinensis, were grown for 8 to 31 days by CPTC following successful preliminary studies with adult pancreas. Adult anolian endocrine pancreas was maintained for up to 7 days by CPTC. The pancreatic explants were examined morphologically by light and electron microscopy. The functional integrity of the endocrine cells was evaluated by measuring hormone levels of the explants and in the basal medium and by determining the kinetics of hormone release. The pancreatic endocrine cells from fetal and adult anoles were functionally and morphologically intact after CPTC. The exocrine pancreas was not maintained during culture. This study demonstrates for the first time the growth of the reptilian endocrine pancreas in culture. The splenic pancreas of the adult green anole, Anolis carolinensis, is rich in endocrine cells and contains about 300 ng of insulin (Rhoten, 1973a) and more than 3,000 ng of glucagon (Rhoten, 1976). The glucagon-containing alpha cells are the predominant cell type, followed in number by somewhat fewer insulin-containing beta cells, still fewer somatostatin-containing delta cells (10%15%), and a small percentage of pancreatic polypeptide-containing F cells (Rhoten, 1973a; Rhoten and Smith, 1978; Rhoten and Hall, 1981). The functional attributes associated with these anolian pancreatic endocrine cells include attenuated insulin secretion, in comparison to mammals, in response to a glucose challenge, a relative insensitivity of the alpha cells to changes in glucose levels, and hyperglycemia (Rhoten, 1973a, 1973b, 1974a, 1978; Marschall and Gist, 1973). Thus, the fetal anolian pancreas may be a useful model with which to explore the cellular mechanisms underlying the development of reduced islet cell sensitivity to glucose and hyperglycemia. An attenuated insulin secretory response to glucose is also found in diabetes mellitus and in fetal beta cells of a number of mammalian species including man (see Rhoten, 1980).Furthermore, hyperglycemia is the sine qua non for the diagnosis of diabetes. Therefore, the morphological and functional differentiation of 0003-276X/82/2031-0165$03.000 1982 Alan R. Liss, Inc. fetal anolian pancreas grown in continuousperifusion tissue culture was examined. Prior to using fetal pancreas, fragments of adult splenic pancreas were grown in continuousperifusion tissue culture (CPTC).Results with the adult pancreas were encouraging and perifusion cultures of 24-day postoviposition splenic pancreases were initiated. The fetal pancreases were grown for up to 31 days by the CPTC method. After 31 days in CPTC, the endocrine cells were intact and alpha, beta, and delta cells could be identified structurally with ease. The presumptive acinar component was nongranulated and appeared to be in various stages of degeneration. The results demonstrate for the first time the growth of the reptilian endocrine pancreas in culture. MATERIALS AND METHODS Adult males and gravid females of Anolis carolinensis were obtained from a biological supply house (The Snake Farm, LaPlace, La). The animals were maintained in an environmental chamber (Calumet Scientific) at a day temperature of 32 f 1"C. The day length (light cycle) was 14 hours and the night length (dark cycle)was 10 hours. The relative humidity was maintained at about 60%. The above condi_ _ _ . ~ Received October 1, 1981; accepted December 23, 1981 166 W.B. RHOTEN tions are consistent with observations on the preferred body temperature and effects of environment on Anolis carolinensis (Licht, 1968, 1971; Crews e t al., 1974). The animals were offered food, instars of Galleria mellonella (the wax moth) or Tenebrio molitor (mealworms), daily. The cages of the gravid females were examined a t least four times daily for eggs. The eggs were incubated on gauze wicks a t 28 rn 2°C wet bulb a s described previously (Rhoten and Hall, 1982). Aseptic technique was used during excision of the pancreas from adult and fetal anoles. The splenic pancreases were rinsed in sterile, Hanks’ balanced salt solution containing penicillin (200 IUiml),streptomycin (200 pggiml) and an antimycotic (5 pgiml). The tissue was cut into small pieces, about 0.5 mm on a side, as required. The tissue was transferred to culture media and injected via a valve into the perifusion chamber of a sterile, filled and operating continuous-perifusion tissue culture (CPTC) system (see below). Fragments of adult splenic pancreas were utilized with two different culture media prior to using fetal splenic pancreas. The medium was either CMRL-1066 or Ham’s F-12 (Gibco),both containing fetal calf serum (lo%), 2 mgiml glucose, and penicillinstreptomycin (100 IUiml and 100 pgiml, respectively). The concentration of glucose selected (2 mgiml) evokes no significant increase in insulin release from perifused splenic islets of adult anoles (Rhoten, 1974a) and is only slightly less than the normal plasma glucose levels of adult anoles (Rhoten, 1973a). Media were changed daily or every other day. Since it is well known that temperature plays an important role in the metabolism of poikilothermic vertebrates like the reptiles, the anolian cultures were kept in an environmental chamber under the conditions described above. In particular, i t should be noted that the temperature regimen included 14 hours a t 32°C and 10 hours a t 24°C. The perifusion system used in this study (CPTC) was developed in the laboratories of Dr. Paul E. Lacy (Lacy et al., 1976). Two basic perifusion systems have been developed: one system requires a carbon dioxide incubator, the other does not. In the latter system, as modified for use in this study, a closed, external “lung” (permitting gas exchange) is required. The lung consists of a 500-ml Erlenmeyer flask with a #7 silicone rubber stopper with multiple inlet and outlet ports for the tissue culture media within Silastic tubing and the gas phase of 7% carbon dioxide-93% air, saturated with water. The tissue fragments were grown on Nitex, a monofilament wire cloth (Tetko, Inc.), in standard perifusion chambers (Swinnex) that are connected by a series of valves and couplers to the culture media. Circulation of media is maintained by an infusion pump (Extracorporeal Medical Specialties, Inc.). The entire assembly was placed in the environmental chamber. The dynamics of insulin and glucagon release from some cultures were determined during shortterm perifusion with Krebs-Ringer-bicarbonate solution of intact explants. The details concerning the perifusion procedures are available elsewhere (Rhoten, 197313; Clements and Rhoten, 1976). Hormone levels were measured by radioimmunoassay (Wright et al., 1968; Heding, 1971). Explants taken for electron microscopy were fixed either in a dilute glutaraldehyde-formaldehyde solution (Karnovsky, 1967)consisting of 3% glutaraldehyde, 2% formaldehyde, 0.09 M sodium cacodylate, and 4.5 mM calcium chloride, or in 2.5% glutaraldehyde in 75 mM phosphate buffer, both a t pH 7.2. The explants were postfixed in 170osmium tetroxide, dehydrated, and embedded in araldite ACM (Fliika, A.G., Switzerland). Thin sections were stained with 2% aqueous uranyl acetate and lead citrate (Reynolds, 1963). Tissue for light microscopy was fixed in 3% glutaraldehydeBouin’s solution and processed routinely for embedding in glycol methacrylate (JB-4, Polysciences, Inc., Warrington, Pa). One part promoter was added to 20-25 parts catalyzed plastic. Thick (1.5-pm) sections were stained with aldehyde fuchsin-trichrome (Epple, 1967). RESULTS Adult pancreas The pancreatic endocrine cells of adult anoles were maintained in the continuous-perifusion tissue culture system in good condition for 7 days when the cultures were terminated (Fig. 1).At least two pancreatic endocrine cell types were seen a t low magnification in aldehyde fuchsin-trichrome (AFT)preparations of the explants after 7 days of CPTC (Fig. 1).The A hhrwia tions ~ ~ ~~ a , acinar cells A. alpha cell H. beta cell CPTC. continuous-perifusion tissue culture D. delta cell e. endocrine cells s. ”small granule” cell PERIFIJSION CULTURE O F ANOLIAN PANCREAS Fig. 1. Adult splenic pancreas grown by CPTC for 7 days in nutrient mixture F-12. The endocrine pancreas is mostly intact, whereas the acinar pancreas is degenerating or has disappeared. Aldehyde fuchsin-trichrome staining of glycol methacrylate-embedded explant. x 112. Fig. 2. Adult splenic pancreas grown by CPTC for 7 days in medium CMRL-1066. Aldehyde fuchsin-trichrome-stained section of glycol methacrylateembedded explant. Endocrine cells make up most of the field with the aldehyde fuchsin-reactive beta cells appearing black. The alpha cells light microscopic appearance of the endocrine cells was similar for cells grown in medium CMRL-1066 (Fig. 2) or in nutrient mixture F-12 (Fig. 1).At higher magnification, the cytoplasm of the endocrine cells was colored in one of three ways with the AFT procedure: blueblack (aldehyde fuchsin stained), reddishorange, or grayish-pink. These tinctorid properties of the CPTC pancreas were typical of those seen in the normal adult anolian pancreas (Fig. 3). Another striking feature of the pancreas maintained in CPTC was the nearly complete absence of readily identifiable exocrine pancreas (Fig. 1 and Fig. 2). The blood vascular spaces, which were characteristic of the endocrine areas in the normal pancreas (Fig. 3), were not preserved in CPTC (Fig. 2). Insulin and glucagon content were similar with the two different culture media: insulin, about 1 2 pgiexplant of splenic pancreas for both media (range of 10.0 to 13.7, n = 4), and glucagon 62 and 120 nglexplant of splenic pan- 167 and the delta cells are gray. The acinar cells are degenerating or have disappeared (compare with Fig. 3). X 280. Fig. 3. Normal adult splenic pancreas. The endocrine cells consist of aldehyde fuchsin-reactive beta cells (black cytoplasm). alpha cells, and delta cells (both with a gray cytoplasm). The acinar cells have relatively large zymogen granules (black)in the apical region. Compare with Figure 2. Aldehyde fuchsin-trichrome-stainedsection of glycol methacrylateembedded tissue. X 280. creas for CMRL-1066 and F-12, respectively (n = 1 each). Insulin was released into the media during CPTC and the levels were similar for the two different media (Fig. 4). At day 7 of CPTC, the insulin release rate was 6.1 ngiday in CMRL-1066 and 10.2 ngiday in F-12. The insulin release rates appeared t o stabilize at 3-4 days of CPTC and showed only a modest decline thereafter (Fig. 4).Since these results indicated that adult beta cells had adapted to the culture conditions by 5 days of CPTC, another set of cultures was initiated to examine the kinetics of hormone release a t day 5 of CPTC. Explants grown in CPTC for 5 days showed enhanced insulin and glucagon secretion when challenged with appropriate secretagogues during perifusion with Krebs-Ringer-bicarbonate solution in vitro (Fig. 5). In response to a low concentration of glucose (0.5 mgiml) and arginine (10 mM), the explants released about 50% more glucagon than in the basal medium (Fig. 5). Insulin secretion was increased when 168 - W.B. RHOTEN 100 T m n M R L-I088 +I ; 80 aQ. Y n 4 80 w -I 40 E -1 a a 20 z 1 2 9 4 5 6 7 1 2 3 4 5 8 Days Fig. 4. Insulin release from the splenic pancreas of Anolis carolinensis during CPTC. The splenic pancreases were maintained in either medium CMRL1066 (left side)or nutrient mixture F-12 (right side). Day 1 of CPTC was used a s 10070and the amount of insulin in the medium on subsequent days was related to day 1. the glucose concentration was raised (Fig. 5). Insulin levels in the perifusate were essentially undetectable before exposure of the explants to a high concentration of glucose (Fig. 5). Glucagon secretion became attenuated during perifusion with the medium containing a high concentration of glucose (Fig. 5). Ultrastructurally, the explants showed variable degrees of cytoplasmic granulation after perifusion (Fig. 6). In summary, the endocrine cells of the adult anolian splenic pancreas were functionally and structurally intact after being grown for up to one week in CPTC. Therefore, the growth of fetal anolian pancreas under similar conditions was attempted. Fetal pancreas Twenty-four-day postoviposition splenic pancreases were grown for 8 to 31 days by CPTC in medium CMRL-1066. After 8 days in CPTC, the endocrine cells were moderately to well granulated (Fig.7). The presence of cells of the acinar pancreas was not apparent. The three major endocrine cell types (alpha, beta, and delta cells) could readily be identified in the electron microscope after 8 days in CPTC (Fig. 7). These endocrine cells resembled closely those present in neonates (postoviposition time to birth was 30.1 + 0.2 days at 28°C wet bulb) and adults, with the exception of a more heterogeneous appearance of the secretory granules in some cells. An additional endocrine-like cell type was occasionally observed. The secretory granules were relatively small, 2 5 30 40 55 70 80 T I M E (mi") Fig. 5. Hormone secretion from adult splenic pancreas grown by CPTC for 5 days. After 5 days of CPTC in medium CMRI,-1066. the chamber containing the explants was disconnected from the culture apparatus and connected t o the short-term perifusion system. The explants were perifused with 1 mg of glucoseiml from 0 t o 28 minutes, and with 10 mM arginine from 28 t o 90 minutes in the presence of either a low concentration of glucose, 0.5 mgiml (28-58 minutes). or a high concentration of glucose, 6 mgiml(58-90 minutes). Insulin release ( 0)was stimulated markedly in the presence of high glucose. Insulin levels in the perifusate were not consistently detectable before exposure t o high glucose. Glucagon secretion (H) was elevated in a low concentration of glucose and reduced in a high concentration of glucose. The dead space of the system was equivalent t o 2 minutes of perifusion. An explant from this perifusion can be seen in Figure 6. electron dense and elliptical in profile in these endocrine-like cells (Fig. 7). The presumptive acinar component lacked zymogen granules. Explant insulin content was 14 ng a t 10 days and had increased to 42 n g at 20 days (mean of two separate perifusion cultures). After 31 days in CPTC, the endocrine cells were intact and alpha, beta, and delta cells could be identified (Fig. 8). Many alpha cells exhibited an unusual degree of electron density differences in their granules (Fig. 8). Some endocrine-like cells could not be identified on the basis of their secretory granule morphologies (Fig. 8). A few cells contained small, elliptic and electron-dense granules (Fig. 8). Insulin secretion, as reflected in insulin levels of basal media, tended to stabilize at low levels after an initial period of decline lasting about 4 days (Fig. 9). Insulin release was about 1nglday per explant at days 8-10 of CPTC and was 0.5 nglday per explant at day 31 in one culture (Fig. 9). Thus, the endocrine cells of fetal anolian pancreas can be maintained structurally and functionally for up to 31 days by CPTC. PERIFUSION CULTURE O F ANOLIAN PANCREAS Fig. 6. Adult splenic pancreas grown by CFTC for 5 days and perifused in vitro. This explant was from the cultures used to obtain the hormone release data in Figure 5. The de- 169 g e e of granulation of the endocrine cells varies considerably. Alpha cells and beta cells are present. X 6,000. were present. The insulin content of cultured fetal anolian pancreas increased more than The anolian endocrine pancreas has been 10-fold from the time CPTC was initiated at grown and maintained long-term in continu- day 24 postoviposition (3.1 i 0.75 nglsplenic ous-perifusion tissue culture (CPTC). Fetal pancreas, n = 3) until 20 days of CPTC had pancreas was grown for up to 31 days by CPTC elapsed (42 ngiexplant). This amount of insulin and morphologically intact endocrine cells is, however, considerably less than the levels of DISCUSSION 170 W.B. RHOTEN Fig. 7. Day 24 postoviposition pancreas grown by CPTC for 8 days. Alpha, beta, and delta cells are identified easily by their morphognomonic secretory granules. A portion of an endocrine-like cell with electron-dense and relatively small secretory granules is present (arrow). Most of t h e endocrine cells are moderately to well granulated. X 3.750. insulin found in the adult splenic pancreas (about 300 ng; Rhoten, 1973a). The glucagon content a t the time of initiation of CPTC, 10.8 + 1.3 nglsplenic pancreas, was 3- to 4-fold greater than that found for insulin, hut explant levels of glucagon were not measured during CPTC. Levels of insulin released into the basal media were quite low during CPTC (about 1 ngiday per explant) hut tended to stabilize a t about day 4 of culture. Cells with alpha granules, which contain glucagon (Rhoten and Hall, 198l), were present in explants after 31 days of CPTC; however, many alpha cells had an unusual degree of electron density differ- PERIFUSION CULTURE OF ANOLIAN PANCREAS 171 Fig. 8. Day 24 postoviposition pancreas grown for 31 days by CPTC. Some of the endocrine-like cells can be identified as alpha or beta cells on the basis of their secretory granule morphologies. An endocrine-likecell with small elec- tron-dense granules can also be seen. The cell type of the other cells present (e)cannot be determined by morphology alone. X 4.500. ences in their granules (see Fig. 8). F cells which contain pancreatic polypeptide (PP) may have been present in the cultures, but they would not have been distinguished with the techniques employed in this study. As reported recently (Rhoten and Hall, 19811, the secretory granules of the PP-containing F cells of the adult anole cannot be identified with certainty, especially with regard to the secretory granules of the D cells, unless immunocytochemical techniques are used. The small, elliptic and electron-dense granules that were occasionally observed in the cultured explants appear to be similar to those found in the endocrine-like cells during the early postoviposition period (Rhoten and Hall, 1982). Cells with these small, electron-dense granule profiles are found rarely in the adult and in the cultured adult pancreas. Granules of similar electron microscopic appearance are found in the early postoviposition pancreas and their contents can be identified by immunocytochemistry (Rhoten and Hall, 19821, but the content of these diminutive granules in the cultured pancreas and in the adult pancreas is not known. The apparent remnants of the acinar component lacked zymogen granules after 31 days of CPTC, suggesting the failure of the acinar cells to continue development during culture since these cells are well granulated in the neonate and in the adult. The nearly complete absence of readily identifiable acinar cells and the numerous vacant areas in the cultured explants of adult splenic pancreas indicate that the adult 172 W.B. RHOTEN 0 DAYS O F CULTURE 15 20 25 2 4 6 8 10 ,’ /I-\,; ; \ o I 2 3 4 5 6 7 8 9 1 0 0 DAYS 30 \ 12 14 16 OF CULTURE Fig. 9. Insulin release into basal medium from fetal splenic pancreas grown by CPTC for up to 31 days. The growth medium used was CMRL-1066 supplemented with fetal calf serum and glucose (see Materials and Methods). Media were changed daily or every other day and insulin levels measured by radioimmunoassay. The number of cultures ( < > I included in the data (n)decreased during CPTC as the cultures were terminated (days 1-10. n = 4; day 11. n = 3; days 12-16, n = 1). Insulin release was determined for only one culture grown for 31 days ( 0 ). Vertical lines indicate the standard error of the mean. exocrine pancreas degenerates and disappears during CPTC (compare Figs. 1 and 2 with Fig. 3). Thus, neither the presumptive acinar component of the fetal pancreas nor the exocrine pancreas of the adult appears to be maintained in CPTC under the present conditions. Disappearance or dedifferentiation of the exocrine component during culture of mammalian pancreas is not uncommon (see Lazarow et al., 1973; Lacy and Gingerich, 1977; Hellerstrom et al., 1979). In contrast, the endocrine component of both the fetal and adult pancreas was preserved in CPTC. The morphological integrity of the endocrine pancreatic cells during CPTC was evident at both the light microscopic and the electron microscopic levels. The functional integrity of the endocrine cells grown in CPTC was demonstrated by the maintenance of, or the increase in, hormone levels during culture, the release of insulin into the basal medium, and the characteristic secretory response of alpha cells and beta cells to conditions that normally are either stimulatory or inhibitory. The finding of stabilized insulin release at 3-4 days of CPTC with adult explants (see Fig. 4) indicates that the beta cells have adapted to the culture environment. I t seems likely that a substantial portion of the insulin release, which occurs at days 1 and 2 of CPTC, reflects the nonphysiologic release of insulin as the cells adapt to culture conditions. If so, then a more meaningful comparison in terms of insulin release is between day 7 of CPTC and day 4.Insulin secretion into basal medium by adult explants at day 7 was about 75% of that at day 4 with either medium CMRL-1066 or nutrient mixture F-12 (Fig. 4). Insulin secretion was enhanced by an elevated concentration of glucose in the presence of arginine as has been found with short-term incubation of anolian splenic pancreas (Rhoten, 1973b, 1973c, 1974a. 197413).Glucagon secretion, on the other hand, was reduced in the high glucose containing medium, and enhanced in a medium with a low concentration of glucose (see Fig. 5). These results are in accord with our prior findings on glucagon secretion by perifused splenic pancreas of the anole (Rhoten, 1978). The results indicate that the anole has additional utility as a model with which to study the regulation of pancreatic endocrine cells in a naturally occurring state of attenuated insulin release and a reduced population of pancreatic beta cells. Such investigations with the anolian pancreas appear to be desirable in efforts to understand similar physiologic and anatomic states related to the etiology and pathogenesis of diabetes mellitus. Furthermore, the growth and differentiation of pancreatic endocrine cells is potentially of great importance in considering the transplantation of islets or individual islet cell types because for transplantation it would be desirable to harvest cells with known secretory and synthetic characteristics and known mitotic potential. Such parameters can be evaluated readily in continuous-perifusion tissue culture. In summary, the splenic pancreas from adult and fetal green anoles has been grown in continuous-perifusion tissue culture. The pancreatic endocrine cells were functionally and morphologically intact after long-term culture (up to 31 days for the fetal explants). The exocrine pancreas virtually disappears during culture. ACKNOWLEDGMENTS Supported in part by NSF grant No. PCM7617997, Public Health Service grant AM19538, and a grant from the New Jersey Affiliate of the American Diabetes Association. The generous gifts of antisera by Drs. Tager and Wright, and the gift of insulin and glucagon by PERIFUSION CULTURE OF ANOLIAN PANCREAS Dr. Root are gratefully acknowledged. The author thanks Marge Pascavage for typing the manuscript. LITERATURE CITED Clements, R.S., Jr., and W.B. Rhoten (1976) Phosphoinositide metabolism and insulin secretion from isolated rat pancreatic islets. J. Clin. Invest., 57:684-691. Crews, D.. J.S. Rosenblatt, and D.S. Lehrman (1974)Effects of unseasonal environmental regime, group presence, group composition and males' physiological state on ovarian recrudescence in the lizard. Endocrinology, 94: 541-547. Epple. A. (1967)A staining sequence for A, B, and D cells of pancreatic islets. Stain. Technol., 42:53-61. Heding. L.G. (1971) Radioimmunological determination of pancreatic and gut glucagon in plasma. Diabetologia. 7: 10-19. Hellerstrom. C.. N.J. Lewis, H. Borg. R. Johnson, and N. Freinkel (1979)Method for large-scale isolation of pancreatic islets by tissue culture of fetal rat pancreas. Diabetes, 28:769-776. Karnovsky. M.J. (1967) The ultrastructural basis of capillary permeability studied with peroxidase a s a tracer. J. Cell Biol.. 35213-236. Lacy, P.E.. E.H. Finke, S.Conant, and S. Naber (1976)Longterm perifusion of isolated rat islets in vitro. Diabetes, 2,5: 484-493. Lacy, P.E., and R.L. Gingerich (1977)Approaches to culturing beta cells. In: Pancreatic Beta Cell Culture. E. van Wasielewski and W.L. Chick, eds. Excerpta Medica. Amsterdam, pp. 37-45. Lazarow. A.. L.J. Wells, A:M. Carpenter, O.D. Hegre, R.J. Leonard, and R.C. McEvoy (1973) Islet differentiation, organ culture, and transplantation. Diabetes, 22r877-912. Licht. P. (1968) Response of the thermal preferendum and heat resistance t o thermal acclimation under different photoperiods in the lizard Anolis carolinensis. Am. Midland Natur., 79:149-158. Licht, P. (1971)Responseof themalereproductivesystem t o 173 interrupted-night photoperiods in the lizard Anolis carolinensis. Z. Physiol.. 73:274-284. Marschall. C., and D.H. Gist (1973)Glycolytic and lipolytic effects of ovine FSH and estradiol-17 beta in the lizard Anolis carolinensis. Gen. Comp. Endocrinol.. 20:407-412. Reynolds, E.S. (1963) The use of lead citrate a t high pH a s an electron opaque stain in electron microscopy. J. Cell Biol., I7:208-212. Rhoten. W.B. (1973a) Insulin content and B cell morphology of the pancreas after chronic insulin treatment in a lizard. J. Exp. Zool., 184:313-320. Rhoten. W.B. (1973b) Perifusion of saurian pancreatic islets and biphasic insulin release following glucose stimulation. Comp. Biochem. Physiol.. 45A:1001-1007. Rhoten, W.B. ( 1 9 7 3 ~Insulin ) release from pancreatic islets of a lizard following leucine or arginine. J. Herpetol., 7: 207-210. Rhoten. W.B. (1974a) Sensitivity of saurian pancreatic is lets to glucose. Am. J. Physiol., 227:993-997. Rhoten. W.B. (1974b) Effects of cytochalasin B and other agents on insulin secretion from saurian islets. Comp. Riochem. Physiol., 47A:959-970. Rhoten, W.B. (1976) Glucagon levels in pancreatic extracts and plasma of the lizard. Am. J. Anat.. 147.131-137. Rhoten. W.B. (1978) Effects of glucose on glucagon secretion by the anolian splenic pancreas. Proc. Soc. Exp. Biol. Med., 257:180-183. Rhoten. W.B. (1980) Insulin secretory dynamics during development of rat pancreas. Am. J . Physiol., 239:E57-E63. Rhoten, W.B., and C.E. Hall 11981) Four hormones in the pancreas of the lizard, Anolis carolinensis. Anat. Rec.. 19: 89-97. Rhoten, W.B., and C.E. Hall (1982) An immunocytochemical study of the cytogenesis of pancreatic endocrine cells in the lizard Anolis carolinensis. Am. J. Anat. 163: 181-193. Rhoten. W.B., and P.H. Smith (1978) Localization of four polypeptide hormones in saurian pancreas. Am. J . Anat., 251:595-601. Wright, P.H.. D.R. Makulu. W.J. Malaisse, N.M. Roberts, and P.-L. Yu (1968) A method for the immunoassay of insulin. Diabetes, 17:534-546.