TECHNICAL NOTE |
Correspondence to: Helmut Wicht, Anatomisches Institut II, Klinikum der Johann Wolfgang Goethe-Universität, D-60590 Frankfurt, Germany. email: wicht@em. uni-frankfurt.de
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording doseresponse curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to ß-adrenergic stimuli inducing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin. Melatonin concentrations were determined by ELISA. cPKA, phosphorylated CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Doseresponse curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Doseresponse curves from immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quantification and localization of biomolecules, and may even be more sensitive than immunoblotting. (J Histochem Cytochem 47:411419, 1999)
Key Words: immunocytochemistry, cell cultures, image analysis, quantification, cytopharmacology, third messengers, phosphorylation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell cultures are widely used to investigate responses of biological systems to different stimuli by recording doseresponse curves of different agonistic and antagonistic agents that affect both secreted and intracellular molecules. Their concentration is usually determined either in the medium or in cell homogenates by RIA (radioimmunoassays), ELISA (enzyme-linked immunosorbent assay), or immunoblotting. Compared to these techniques, analyses of immunocytochemical preparations appear to be promising alternatives to determine the level of intracellular molecules because they allow simultaneous semiquantification and localization of the molecules and require much less biological material. We have therefore developed a measurement routine that provides semiquantitative data on the concentration and distribution of intracellular proteins or peptides in immunocytochemical preparations.
The method is based on image analysis and its development included the following steps: (a) determination of a measurement parameter that correlates with the amount of antigen in immunoblots; (b) determination of this parameter in immunocytochemical preparations; and (c) validation of the measurements in immunocytochemical preparations by comparing dosereponse curves recorded from immunoblots and immunocytochemical preparations.
The method was developed using a well-characterized cell culture system, i.e., isolated rat pinealocytes (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals, Preparation of Rat Pinealocyte Cultures, and Stimulation
All experiments were conducted in accordance with guidelines on the care of experimental animals as approved by the European Communities Council Directive (86/609/EEC). For cell culture, pineal glands were removed from adult male Wistar rats. For isolation of pinealocytes, the glands were immediately dissociated by papain digestion and repeated pipetting as previously described (
Immunoblotting
After stimulation, the medium was removed and used for ELISA (see below). The cells were lysed directly on the coverslips by 50 µl of sample buffer according to
|
The membranes were incubated with polyclonal antibodies detecting Ser-133 phosphorylated CREB (pCREB; New England Biolabs, Beverley, MA; diluted 1:3000) (
Immunocytochemistry of Cell Cultures
Pinealocytes were cultured for 13 days and stimulated as indicated. After stimulation, the medium was collected to determine the melatonin concentration by ELISA. The cells were fixed on the coverslips with 4% paraformaldehyde in PBS for 10 min and washed in PBS. To block endogenous peroxidase, the preparations were treated with methanol containing 0.45% hydrogen peroxide for 10 min. They were then washed and preincubated with 10% normal goat serum (30 min), and incubated with polyclonal antibodies against pCREB (1:500; New England BioLabs) or ICER (1:70,000) (
ELISA
Medium was collected from each cell culture 6 hr after the beginning of the stimulation. Melatonin concentration was measured by a commercial ELISA using biotinylated melatonin (IHF GmbH; Hamburg, Germany) as tracer. The detection limit of this assay is 2 pg melatonin/ml. The intra-assay and interassay variation coefficients were below 15%.
Statistical Analysis
Apart from single immunoblots, the graphs show the means (±SEM) of 320 measurements which stem from one of three experimental series. These data were subjected to Student's t-test or one-way ANOVA with subsequent Bonferroni tests for multiple comparisons using the program PRISM (GraphPad; San Diego, CA) with p0.05 as the criterion of significance.
Optical Equipment/Computer Hardware
The images were recorded with a standard black/white CCD camera equipped with a macro-objective for analysis of immunoblots. The blots were placed in a dark chamber to reduce stray light and were illuminated either with transmitted light (chemoluminescence blots on photographic films; Figure 1A and Figure B) or with reflected light (DAB-reacted blots on nitrocellulose membranes; Figure 1C). For analysis of the immunocytochemical preparations, the camera was connected to a microscope (Zeiss, Photomikroskop II; Oberkochen, Germany). The light source was controlled by a finely adjustable constant voltage transformer (Zeiss). A continuous color filter (400700 nm; Zeiss) was used to adjust the contrast (see
Digitization of Images
All images were digitized at a spatial resolution of 512 x 512 pixels and a gray value resolution of 8 bits (gray values 0255). The present method requires controlled and reproducible access to the parameters of the analogue/digital (intensity/gray value) conversion. In the VIDAS 2.1 system, this conversion is controlled numerically via the software, but not all systems allow this control (
Measurement of Area and Intensity of Signals in Immunoblots
Individual signals in immunoblots vary in size and intensity. On the basis of the assumption that the antigen amount affects both variables, we considered the integrated intensity of the signals a suitable measurement parameter to estimate the antigen amount. The integrated density can be determined by summing up the gray values of all pixels representing an immunosignal. To examine the correlation between this parameter and the antigen amount, we analyzed calibrated immunoblots containing known antigen amounts in the different lanes (Figure 1). To cover the entire dynamic range of gray values (0255), the analogue/digital conversion was set in such a way that pixels of a gray value close to 0 were attributed to the most intensely immunostained areas (red range in Figure 1D and Figure 1F) and pixels of gray values close to 255 to the unstained background (green range in Figure 1D and Figure 1F). The image was then inverted (negative image in Figure 1E) to attribute the numerically highest gray values to pixels that represent the most immunoreactive sites. Thereafter, the sum of gray values of all pixels belonging to an individual band in the blot was calculated. This value was called SUMDENS (for sum of densities). To normalize the means of SUMDENS values from different blots, each value was given as percentage of the maximal value of the corresponding curve. The normalized means were plotted against the logarithm of the antigen concentration.
Measurement of Area and Intensity of Signals in Immunocytochemical Preparations
Each experiment required a set of 2030 coverslips loaded with pineal cells that were prepared on the same day, cultured for the same time period, and treated with various concentrations of the stimulants. Typically, three to five coverslips were treated with the same concentration. The cells were then fixed and immunoreacted in one batch, i.e., they were exposed to identical antibody solutions, buffers, and chromogens, for exactly the same amount of time.
Compared to the analysis of the immunoblots, the semiquantitative analysis of immunocytochemical preparations raises the following additional problems. (a) Whereas in immunoblots several different lanes can be digitized as a single image, the images from several immunoreacted coverslips must be digitized separately but under identical conditions. (b) The cellular or subcellular compartments containing the immunoreactivity need to be identified and separated from their surroundings. (c) The number and density of cells per coverslip varies, and this variation needs to be corrected to obtain comparable measurements. Finally, (d) in different experiments comprising different sets of coverslips, the intensity of the immunocytochemical reaction may vary from set to set and has to be normalized.
From each set of coverslips, the preparation with the most intense immunoreactivity (Figure 2A, upper panel) was used to fix the settings of the light intensity, the color filter, and the analogue/digital conversion. These settings were visually controlled by a pseudo-color mask (Figure 2A and Figure 2B), which was displayed online. The adjustable color filter was used to fit the contrast of strongly or weakly immunoreactive coverslips into the computer's dynamic range. After these settings were determined for the most intensely stained preparation of one experiment, they were kept constant during the digitization of all other less intensely immunoreactive preparations from the same experiment (Figure 2A, lower panels). During these digitizations, only very small changes in the intensity of the light source were allowed to adjust the background of each preparation to exactly 255. Therefore, the effects of variations in, e.g., the ambient light, intensity fluctuations of the light bulb, and differing thicknesses of the glass slides could be eliminated. Depending on the density of the cells, approximately 50200 cells were contained in one image. Typically, three or four digital images were created from different regions of one coverslip and three to five coverslips were used for one particular stimulus concentration. Therefore, one data point reflects measurements of 5004000 cells. This digitization procedure allowed each individual experiment to be normalized to the maximal/minimal response that was obtained under the particular conditions of the experiment itself.
|
The procedures to determine the SUMDENS in an individual digital image and to correct it for various cell numbers are illustrated in Figure 2. The first image of an image analytical sequence (Figure 2B, still displaying the pseudo-color mask from the digitization) was inverted to generate a measurement image (not shown) in which strongly immunoreactive sites are represented by pixels with numerically high gray values. The first image was then slightly contrast-enhanced (not shown) and segmented to generate two binary images, one representing the immunoreactive area (Figure 2C), and the other representing the total area of all cells (Figure 2D). The segmentation thresholds (arrows in Figure 2B) were kept constant for all images stemming from one set of coverslips. One binary image (Figure 2C) served as a mask to determine the SUMDENS value with reference to the inverted image; the other (Figure 2D) was used to determine the total area covered by cells. The SUMDENS value was divided by this area value, and the resulting parameter was called corrected SUMDENS (corr. SUMDENS, for cell number-corrected sum of densities).
Comparison of Results from Immunoblots and Immunocytochemistry
To validate the recordings from immunocytochemical preparations, we subjected paired sets of coverslips to identical treatment and processed one set for immunocytochemistry and the other for immunoblotting. The SUMDENS and corrected SUMDENS, respectively, were measured in the blot and on the coverslips and the resulting doseresponse curves were compared.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calibration Measurements in Immunoblots
SUMDENS values were measured in several calibrated immunoblots that contained known amounts of antigen in each lane detected by chemoluminescence. The measurements provided an array of saturation curves (Figure 3A) for the SUMDENS vs the antigen concentration for all antigens (CREB, pCREB, c-PKA) and exposure times (115 min) tested. A plot of normalized SUMDENS values against the logarithm of the antigen concentration (Figure 3B) showed an almost linear relation between the two parameters. A linear relation was also observed in blots developed with DAB, which is routinely used as a chromogen for immunocytochemical preparations. When the immunoreaction for c-PKA was visualized in the same preparation, first by chemoluminescence (on film, Figure 1B) and then with DAB (directly on the nitrocellulose membrane, Figure 1C), the curves reflecting the SUMDENS vs antigen concentration were strictly parallel, including a deviation from linearity at the second lowest antigen concentration detectable (Figure 3C).
|
Comparison of Measurements from Immunoblots and Immunocytochemical Preparations
Treatment of pinealocytes with increasing concentrations of the AMP analogue Sp-DClcBIMPS resulted in an induction of CREB phosphorylation, which was semiquantified in one and the same experiment by both immunocytochemistry (images of the cells are shown in Figure 2A) and immunoblotting (blot is shown in inset of Figure 4A). The immunocytochemical preparations showed the typical nuclear localization of pCREB. The doseresponse curves generated by both methods matched very closely (Figure 4A) and revealed half-effective concentrations (EC50) of 5 or 6 µM in the immunocytochemical preparations or the immunoblot, respectively. At very low concentrations of the stimulant, the measurement of the corrected SUMDENS in the immunocytochemical preparations appeared to be even more sensitive than the SUMDENS measurements in the immunoblot.
|
Reproducibility of Measurements in Immunocytochemical Preparations
The effects of various cAMP agonists and antagonists on the intracellular concentration of pCREB and ICER were measured in a number of immunocytochem-ical experiments. Reproducible and comparable doseresponse curves were obtained from different experimental sets of coverslips that were prepared from different animals on different days. An example is given in Figure 4B, showing corrected SUMDENS values from two such experiments. The cells were stimulated with the cAMP analogue MBCcAMP and immunostained for pCREB; the doseresponse curves obtained in both experiments are almost identical.
Application of the Method in Cytopharmacological Experiments
The intranuclear concentrations of pCREB and the inhibitory transcription factor ICER, as well as melatonin biosynthesis, are affected by ß-adrenergic stimulation in a dose-dependent manner. The doseresponse curves for both melatonin and ICER reached saturation at norepinephrine (NE) concentrations between 0.001 and 0.01 µM, whereas the values for pCREB did not reach saturation even at the highest NE concentration tested (Figure 4C).
Blockade of PKA with the cAMP antagonist Rp-8-CPT-cAMPS reduced the nuclear amount of pCREB (determined via immunocytochemistry) and suppressed induction of melatonin biosynthesis in a dose-dependent manner (Figure 4D).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many immunocytochemical reactions and interactions are nonstoichiometric (
The present study expands the possibilities of the combined immunocytochemical and image analytical approach to routine cytopharmacological applications: the determination of doseresponse curves and the comparison of the effectiveness of various agonists and antagonists on intracellular molecules in cell culture systems. The measurement parameters (SUMDENS and corrected SUMDENS) are integrals of the intensity of an immunosignal over its area; both of these variables contain information on the antigen content (
The required equipment is present in many laboratories, and the image analytical procedure is based on a number of standard operations (segmentations and measurements) that can, in principle, be performed with any scientific image analysis software. The only really critical point is the numerical control of image acquisition, analogue/digital conversion, and segmentation. These parameters must be controlled interactively by the programmer/experimenter and must not be left to automatic adjustments of the computer's or camera's internal soft- or hardware (like automated adjustments of image brightness and contrast). Popular commercial programs, such as Adobe Photoshop or Coral Photo House, usually rely on external program/hardware modules for image aquisition, which may be more difficult to control than the internal modules of scientific software. In addition, they usually do not offer the routines that are required for densitometric and planimetric measurements.
The use of cell cultures and the segmentation step in the measurement routine offer the opportunity for regionalized measurements of antigen content. Images such as the ones shown in Figure 2C and Figure 2D allow measurements in different cell compartments, e.g., cytoplasm or nucleus, thus tracing possible translocations of an antigen from one cell compartment into another. Alternatively, such images can be used to identify subpopulations of cells that show different responses to a given stimulus.
With this method, we identified protein kinases of Type A as catalysts of NE-induced/cAMP-mediated CREB phosphorylation in rat pinealocytes, as is the case in bovine (
In summary, we have presented a method that we have used in our everyday laboratory work and that has proved to be a useful and flexible tool for different kinds of cytopharmacological investigations in cell cultures.
![]() |
Footnotes |
---|
1 The first two authors contributed equally to this study.
![]() |
Acknowledgments |
---|
Supported by the Deutsche Forschungsgemeinschaft, Son-derforschungsbereich 269, Projekt B2.
Jörg Stehle and Martina Pfeffer contributed the data on ICER immunoreactivity to this study; Frank Nürnberger helped with some optical equipment that was needed to make the method work. We thank H.-G. Genieser (Bremen) for the gift of MBC-cAMP, C.A. Molina (New Jersey) for the ICER-antibody, G. Schwoch (Göttingen) for the cPKA antibody, and J. Pfeilschifter (Frankfurt) for his perspective and guidance in the preparation of this manuscript.
Received for publication August 3, 1998; accepted October 28, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agnati LF, Fuxe K, Benfenati F, Zini I, Zoli M, Fabri L, Härfstrand A (1984) I. Methodological aspects. In Agnati LF, Fuxe K, eds. Computer-assisted Morphometry and Microdensitometry of Transmitter-identified Neurons with Special Reference to the Mesostriatal Dopamine Pathway. Acta Physiol Scand Suppl 532:6-36
Baler R, Covington S, Klein DC (1997) The rat arylalkamine N-acetyltransferase gene promotercAMP activation via a cAMP-responsive element-CCAAT complex. J Biol Chem 272:6979-6985
Borjigin J, Wang MM, Snyder SH (1995) Diurnal variation in mRNA encoding serotonin N-acetytransferase in the pineal gland. Nature 378:783-785[Medline]
Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254[Medline]
Coon SL, Roseboom PH, Baler R, Weller JL, Namboodiri MAA, Koonin EV, Klein DC (1995) Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis. Science 270:1681-1683[Abstract]
Cowen T, Thrasivoulou C (1992) A microscopical assay using a densitometric application of image analysis to quantify neurotransmitter dynamics. J Neurosci Methods 45:107-116[Medline]
DobadoBerrios PM, RuizNavarro A, Torronteras R, GarciaNavarro F (1992) Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting. J Histochem Cytochem 40:1715-1724
Foulkes NS, Borjigin J, Snyder SH, SassoneCorsi P (1996) Transcriptional control of circadian hormone synthesis via the CREM feedback loop. Proc Natl Acad Sci USA 93:14140-14145
Geisler JP, Geisler HE, Wiemann MC, Givens SS, Zhou Z, Miller GA (1997) Quantification of p53 in epithelial ovarian cancer. Gynecol Oncol 66:435-438[Medline]
Genieser H-G, Winkler E, Butt E, Zorn M, Schulz S, Iwitzki F, Störmann R, Jastorff B, Døskeland SO, Øgreid D, Ruchard S, Lanotte M (1992) Derivatives of 1-ß-D-ribofuranosylbenzimidazole 3',5'-phosphate that mimic the actions of adenosine 3',5'-phosphate (cAMP) and guanosine 3',5'-phosphate (cGMP). Carbohydr Res 234:217-235[Medline]
Gjertsen BT, Mellgren G, Otten A, Maronde E, Genieser H-G, Jastorff B, Vintermyr OK, McKnight GS, Døskeland SO (1995) Novel Rp-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. J Biol Chem 270:20599-20607
Gonzalez GA, Montminy MR (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675-680[Medline]
Klein DC, Roseboom PH, Coon SL (1996) New light is shining on the melatonin rhythm enzymethe first postcloning view. Trends Endocrinol Metab 7:106-112
Korf H-W, Oksche A, Ekström P, Zigler JS, Gery I, Klein DC (1986) Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum. Science 231:735-737[Medline]
Korf H-W, Schomerus C, Maronde E, Stehle JH (1996) Signal transduction in the rat pineal organ: Ca2+, pCREB, and ICER. Naturwissenschaften 83:535-543[Medline]
KumarSingh S, Vermeulen PB, Weyler J, Segers K, Weyn B, van Daele A, Dirix LJ, van Oosterrom AT, van Marck E (1997) Evaluation of tumor angiogenesis as a prognostic marker in malignant mesothelioma. J Pathol 182:211-216[Medline]
Lehr HA, Mankoff DA, Corwin D, Santeusiano G, Gown AM (1997) Application of a Photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem 45:1559-1565
Maronde E, Middendorff R, Telgmann R, Taskén K, Hemmings B, Müller D, Olcese J (1997a) Melatonin synthesis in the bovine pineal gland is regulated by cyclic AMP-dependent protein kinase type II. J Neurochem 68:770-777[Medline]
Maronde E, Pfeffer M, von Gall C, Schomerus C, Dehghani F, Kroeber S, Wicht H, Taskén K, Olcese J, Stehle J, Korf H-W (in press) Signal transduction in the rodent pineal organ. In Olcese J, ed. Melatonin After Four Decades
Maronde E, Schomerus C, Stehle JH, Korf H-W (1997b) Control of CREB phosphorylation and its role for induction of melatonin synthesis in rat pinealocytes. Biol Cell 89:505-511[Medline]
Øgreid D, Ekanger R, Suva RH, Miller JP, Sturm P, Corbin JD, Døskeland SO (1985) Activation of protein kinase isozymes by cyclic nucleotide analogs used singly or in combinationprinciples for optimizing the isozyme specificity of analog combinations. Eur J Biochem 150:219-227[Abstract]
PerettiRenucci R, Feuerstein C, Manier M, Lorimier P, Savasta M, Thibault J, Mons N, Geffard M (1991) Quantitative image analysis with densitometry for immunohistochemistry and autoradiography of receptor binding sitesmethodological considerations. J Neurosci Res 28:583-600[Medline]
Rittenhouse J, Marcus F (1983) Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium-dodecyl sulfate-containing buffers. Anal Biochem 138:442-448
Roseboom PH, Klein DC (1995) Norepinephrine stimulation of pineal cyclic AMP response element-binding protein phosphorylation: primary role of a ß-adrenergic receptor/cyclic AMP mechanism. Mol Pharmacol 47:439-449[Abstract]
Roseboom PH, Coon SL, Baler R, McCune SK, Weller JL, Klein DC (1996) Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleotide acid in the rat pineal gland. Endocrinology 137:3033-3044[Abstract]
Sandberg M, Butt E, Nolte C, Fischer L, Halbrügge M, Beltman J, Jahnsen T, Genieser H-G, Jastorff B, Walter U (1991) Characterisation of Sp-5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole-3',5'-mono-phosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells. Biochem J 279:521-527[Medline]
Schomerus C, Maronde E, Laedtke E, Korf H-W (1996) Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce phosphorylation of the transcription factor CREB in subpopulations of rat pinealocytes: immunocytochemical and immunochemical evidence. Cell Tissue Res 286:305-313[Medline]
Schwoch G, Hamann A, Hilz H (1980) Antiserum against the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase. Biochem J 192:223-230[Medline]
Stehle JH (1995) Pineal gene expression: dawn in a dark matter. J Pineal Res 18:179-190[Medline]
Stehle JH, Foulkes NS, Molina CA, Simmoneau V, Pevet P, SassoneCorsi P (1993) Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature 356:314-320
Stehle JH, Foulkes NS, Pevet P, SassoneCorsi P (1995) Developmental maturation of pineal gland function: synchronized CREM inducibility and adrenergic stimulation. Mol Endocrinol 9:706-716[Abstract]
Szewczyk B, Kozloff LM (1985) A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Anal Biochem 150:403-407[Medline]
Tamotsu S, Schomerus C, Stehle JH, Roseboom PH, Korf H-W (1995) Norepinephrine-induced phosphorylation of the transcription factor CREB in isolated rat pinealocytes: an immunocytochemical study. Cell Tissue Res 282:219-226[Medline]
Thal DR, Horn M, Schlote W (1995) Selective quantitative analysis of the intensity of immunohistochemical reactions. Acta Histochem 97:203-211[Medline]
Vandesande F (1979) A critical review of immunocytochemical methods for light microscopy. J Neurosci Methods 1:3-23[Medline]
Vigo J, Salmon JM, Lahmy S, Viallet P (1991) Fluorescent image cytometry: from qualitative to quantitative measurements. Anal Cell Pathol 3:145-165[Medline]
Zatz M, O'Dea RF (1976) Regulation of protein kinase in rat pineal gland: increased Vmax in supersensitive glands. J Cycl Nucleotide Res 2:427-439[Medline]