Proc. Nat. Acad. Sci. USAVol. 72, No. 6, pp. 2022-2026, June 1975

Immunohistochemical Localization of 3': 5 '-Cyclic AMP and 3': 5 '-Cyclic GMPin Rat Liver, Intestine, and Testis

(nuclear cyclic GMP/immunocytochemistry)

SHU-HUI ONG, THOMAS H. WHITLEY, NORMA W. STOWE, AND ALTON L. STEINER*

Departments of Medicine and Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, N.C. 27514

Communicated by Ernest L. Eliel, February 28, 1976

ABSTRACT Cyclic GMP and cyclic AMP have been lo-calized in rat liver, small intestine, and testis by a fluores-cent immunocytochemical procedure. In liver, cyclic AMPis distributed along sinusoids predominantly, and in-creased fluorescence is seen in sinusoidal areas after gluca-gon administration. Cyclic GMP is located in nuclear ele-ments and on the plasma membranes of hepatocytes. Injejunum, cyclic AMP is found predominantly at the basaland lateral sides of brush border cells and in the laminapropria, while cyclic GMP is located to the brush bordermembrane, smooth muscle, and nuclear elements. In tes-tis, cyclic AMP is found in cytoplasm of cells at the perim-eter of the seminiferrous tubules and in interstitial cells,while cyclic GMP is visualized on the plasma membrane ofthe cells lining the tubules. Cyclic GMP is also seen onchromosomes of premeiotic spermatocytes and in sperm.These data provide histological evidence implicating di-verse roles for the nucleotides in these tissues. The nuclearlocalization of cyclic GMP in all of these tissues suggests arole for the nucleotide in nucleus-directed events.

Accumulating evidence suggests that cyclic GMP is involvedin a number of cellular events ranging from the action ofacetylcholine at muscarinic receptors (1-3) to growth regula-tion in lymphocytes (4) and fibroblasts (5). In these studiesincreased tissue concentrations of cyclic GMP have beendemonstrated after hormonal stimulation. Despite this evi-dence, the role of cyclic GMP in cell function has been difficultto determine, partly because exogenously administered cyclicGMP in most tissues acts as a weaker cyclic AMP (6).To gain insight into the relative roles of cyclic AMP and

cyclic GMP in cell function, we have recently applied thetechnique of cyclic nucleotide immunocytochemistry tostudies in canine thyroid tissue, and contrasted the localizationof cyclic GMP with that of cyclic AMP. We utilized caninethyroid tissue for these studies because thyrotrophin stimu-lating hormone increases cyclic AMP, without affecting cyclicGMP levels, and cholinergic compounds increase cyclic GMPwhile cyclic AMP is unchanged. We found cyclic GMP to belocated in the area of the follicular plasma membrane border-ing the colloid, and showed an increase in cytoplasmic fluo-rescence after acetylcholine treatment. In contrast, cyclicAMP was found throughout the cytoplasm of the follicularcells, and increased cytoplasmic fluorescence was seen afteradministration of thyrotrophin stimulating hormone. Thedistinct differences in localization of cyclic AMP and cyclicGMP suggested that the specific roles and the intracellularlocalization of these two nucleotides may be related (7).

We have continued to apply this immunocytochemical pro-cedure for the localization of cyclic AMP and cyclic GMP in avariety of rat tissues. In general, the two nucleotides havedistinctly different staining patterns in individual cells.Cyclic AMP is found mostly in cytoplasm and sometimes onplasma membranes, but cyclic GMP is usually distinctlylocalized, most commonly in plasma membranes and nuclearelements. Since this methodology is applied to unfixed tissues,we are most likely detecting cyclic nucleotide bound to recep-tor sites. These studies then provide clues for the location ofreceptors for cyclic AMP and cyclic GMP, which in turnsuggest separate roles for the nucleotides, particularly cyclicGMP in cell function.

MATERIALS AND METHODS

Preparation of Tissue Samples. Fed male Sprague-Dawleyrats, weighing 180-200 g, were utilized in these studies. Intactanimals were killed by blunt trauma in order to minimizestress and pieces of liver, jejunum, and testis were frozen im-mediately in an aluminum foil boat filled with Optimal Cut-ting Temperature Compound (Ames Co., Division Miles Labo-ratories, Inc., Elkhart, Ind.) by immersion in ice-cold acetone.Hypophysectomized rats, purchased from Hormone AssayLaboratories, Chicago, Ill., were sacrificed by cervical dis-location on the tenth day after hypophysectomy and sam-ples of testicular tissue were frozen as described above.

Materials. Antibodies to cyclic nucleotides were preparedas described elsewhere (8). Rabbit gamma globulins andfluorescein-labeled goat antibodies to rabbit IgG (lot 16)were obtained from Miles Laboratories, Inc., Miles ResearchDivision (Kankakee, Ill.). Crystalline glucagon (lot 62C-2450)was obtained from Sigma Chemical Co., St. Louis, Mo. andwas dissolved in 1% albumin-0.9% saline for intraperitonealadministration. Control animals received an equal quantity ofthe vehicle solution intraperitoneally.

Histochemical Procedure. Histochemical localization (9) ofboth cyclic nucleotides was determined by an immunofluo-rescent procedure on frozen tissue sections 4-6 ,m in thicknesswith the use of highly specific immunoglobulin IgG fromrabbit antisera to either cyclic AMP or cyclic GMP as pre-pared by the method of Steiner et al. (8). The frozen sectionswere dried in air on slides and treated in sequence for 30 minwith each of the following: antibody to nucleotide (1:10dilution of antibody to cyclic AMP and 1: 8 dilution ofantibody to cyclic GMP or 1:10 control serum); phosphate-buffered saline (PBS); fluorescein-labeled goat antiserum torabbit IgG in a 1:8 dilution; and PBS. The slides were

2022

Abbreviations: cyclic GMP, guanosine 3':5'-cyclic monophos-phate; cyclic AMP, adenosine 3': 5'-cyclic monophosphate.* To whom reprint requests should be addressed at the Depart-ment of Medicine.

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Immunohistochemical Localization of cAMP and cGMP 2023

s* .; w C ,. . >.*....

z v iS ZW;t I* x

>h=eSlMw

kid idsy* f.s ..,# .. Y.. .S ...

IC Id * .FIG. 1. Immunofluorescent localization of cyclic AMP (a) and cyclic GMP (b) in rat liver (X 120). (c) is a photograph illustrating non-

specific fluorescence. In (d), the cyclic GMP antibody has been incubated with cyclic GMP (5 /AM) prior to staining.

mounted with 50% glycerin in PBS. To demonstrate thespecificity of the procedure, we found that similarly preparedantisera from unimmunized rabbits failed to produce signifi-cant staining. Furthermore, when the antiserum was incu-bated overnight at 40 with the corresponding cyclic nucleotideat 5 MAM, the staining pattern was no longer present, indi-cating competitive inhibition of cyclic nucleotide binding.Incubation with 50-100 fold higher concentrations of theother cyclic nucleotide were required to eliminate the stainingpattern (9). When the antibodies were incubated with ATPor 5'-AMP (10 MM) staining was not eliminated.

RESULTSWe have compared the distribution of cyclic GMP and cyclicAMP in three rat tissues.

Liver. In Fig. 1 is shown the distribution of cyclic AMP andcyclic GMP in liver from a control rat. Cyclic AMP is dis-tributed predominantly along sinusoids (Fig. la). Both plasmamembranes and cytoplasmic elements fluoresce brightly.Perinuclear staining is seen also. While the sinusoidal stainingis most likely of hepatocytes, we cannot eliminate the possi-bility that the fluorescence includes elements of the reticulo-endothelial system. Intranuclear elements do not fluoresce.In contrast, cyclic GMP is localized predominantly in nucleiand plasma membranes of the hepatocytes (Fig. lb). Theplasma membranes of each parenchymal cell fluoresce, out-lining individual cells. There is no outlining of sinusoidal areasand fluorescence in cytoplasm is minimal. Nucleoli fluorescebrightly and in some nuclei a clumped chromatin pattern isseen. When the cyclic GMP antiserum is incubated with 5,uMcyclic GMP, both plasma membrane and nuclear fluorescenceare eliminated (Fig. id). A control with serum from unim-munized rabbits is shown in Fig. lc.

Glucagon has been shown to increase cyclic AMIP levels inliver and increases the release of the nucleotide into the extra-

FIG. 2. Dark field fluorescence micrographs of rat liver, illus-trating the relative intensity of fluorescent staining for cyclicAMP in liver from rats injected with glucagon (a) or from a con-trol animal (b) (X86.5). The exposure times for the photographswere identical. At the bottom of (b) is fluorescent staining in thearea of a central vein.

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FIG. 3. Immunofluorescent localization of cyclic AMP and cyclic GMP in jejunum of the rat (X 120). (a) Cyclic GMP is localized onthe brush border membrane and nucleus in this longitudinal section. (b) is an areafrom the junction of the mucosal and smooth muscleareas (tangential section). In (c) and (d) the sections have been stained with the cyclic AMP antiserum. (c) is a longitudinal section while(d) is a cross section from the crypts area. The arrows point to fluorescence at the lateral side of the brush border cells. Note the minimalfluorescence in (c) and (d) in the area of the brush border membranes.

cellular fluid during liver perfusion (10). Rats were injectedwith glucagon, 1 mg, and the liver was frozen 10 min later.Cyclic AMP levels were measured by radioimmunoassay andincreased 4-fold in the glucagon-treated rats. In the animalstreated with glucagon (Fig. 2a), there was a marked increasein fluorescence compared to liver from a control rat (Fig. 2b).The increased fluorescence in the glucagon-treated animals isconcentrated along sinusoids and membrane fluorescenceappears to increase.

Small Intestine. The distributions of cyclic GMP and cyclicAMP in rat small intestine are distinctly different. CyclicGMP is localized in the villus brush border membrane withminimal staining for cyclic GMP in other areas of the villustip (Fig. 3a). This brush border localization of cyclic GMP isvirtually eliminated when the cyclic GMP antibody is incu-bated with 5 uM cyclic GMP. Cyclic GMP is also found innuclei, especially of cells in the crypts areas. In many cells,the nucleoli fluoresce (Fig. 3b). Smooth muscle also fluorescesbrightly (Fig. 3b).

Cyclic AMP is localized in the lateral and basal sides of thecells of the villus tip (Fig. 3c and d). Minimal staining isfound in the brush border area. This localization of cyclic AMPis consistent with the distribution of adenylate cyclase frommucosal epithelial cells of rabbit intestine (11). Cyclic AMP

is also found in the laminal propria (Fig. 3c) and in smoothmuscle (photograph not shown). Nuclear fluorescence withthe cyclic AMP antibody is minimal.

Testis. In Fig. 4a the distribution of cyclic AMP is shownwithin seminiferous tubules and the interstitial area of amature rat. Prominent staining is found in the cytoplasm andmembranes of the cells on the perimeter of the tubule. Thesecells include both Sertoli and germ cells. Interstitial cells alsofluoresce brightly. Cells in the center of the tubules show onlyminimal fluorescence (photograph not shown). Sperm fluo-resce also.

In contrast, cyclic GMP is found predominantly on mem-branes, but not in the cytoplasm, of cells bordering the tubularmembrane (Fig. 4b). In primary spermatocytes prior tomeiosis chromosomes fluoresce brightly. The specificity of thischromosomal localization of cyclic GMP in this particular cellpool is demonstrated by the absence of chromosomal stainingin the cells bordering the peritubular membrane and in cellsin the center of the tubule that have completed meiosis.When the cyclic GMP antibody is incubated with 5 ,M cyclicGMP, chromosomal staining disappears, but fluorescenceremains when the antibody is incubated with 50-fold greaterconcentrations of cyclic AMP. Incubation of the antiserumwith other nucleotides such as ATP and GTP at significantly

Proc. Nat. Acad. Sci. USA 72 (1975)

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Immunohistochemical Localization of cAMP and cGMP 2025

FIG. 4. Immunofluorescent localization of cyclic AMP and cyclic GMP in rat testis. All views are in cross section. In (a) fluorescence isseen in three seminiferous tubules and an interstitial area stained with the cyclic AMP antiserum (X 120). In (b) the sections have beenstained for cyclic GMP (X 120). Note the fluorescence on membranes of the cells lining the tubule, chromosomal fluorescence, and the ab-sence of fluorescence in the center of the tubule. Insert shows the fluorescence of the premeiotic chromosomes (X 180). (c) Testis from a rathypophysectomized 10 days previously and stained for cyclic GMP (X 120). Note nuclear fluorescence, but not on condensed chromo-somes as in (b). (d) is a lower power view stained for cyclic GMP (X80). Note fluorescence in sperm.

higher concentrations does not eliminate fluorescence. Inaddition, in animals hypophysectomized for 10 days, chro-mosomal localization of cyclic GMP is not seen, but cyclicGMP is seen in nuclear elements in a clumped chromatinpattern (Fig. 4c). Cyclic GMP is also found in sperm frommature animals (Fig. 4d).

DISCUSSION

The demonstration by this immunocytochemical method thatcyclic AMP and cyclic GMP are uniquely distributed in avariety of rat tissues helps to elucidate the roles of the twonucleotides in cell function. The observation that cyclic GMPis found on the plasma membrane of a number of tissues,including canine thyroid (7), liver, small intestinal brushborder, and testicular cells, suggests that the nucleotide isinvolved in membrane function. Since this immunocyto-chemical procedure is performed on unfixed tissue, the freecyclic nucleotides should be lost during the staining proce-dure and only those nucleotides bound to cellular receptorsshould be depicted. Our observation of plasma membranestaining for cyclic GMP thus suggests the presence of bindingsites for cyclic GMP in plasma membranes in a variety oftissues.

It is important to emphasize that this immunocytochemicaltechnique depends upon the recognition by the antibodies of

specific antigenic determinants for the cyclic nucleotides intissue. These determinants most likely include the 3':5'cyclic ring and specific substitutions on the purine nucleus.Fluorescent staining indicates available sites for antibodyrecognition, while the absence of staining is consistent withdeterminants that are unavailable to the immunological re-agents. Thus, it is possible that either cyclic nucleotide mightbe present at specific cellular sites and not be recognized byantibody. Our experience with a number of cyclic GMP andcyclic AMP antisera from different rabbits is that the stainingpatterns are consistent for each cyclic nucleotide. Since eachantiserum most likely contains a number of antibodies withdifferent affinities for the cyclic nucleotide, this consistency ofstaining pattern decreases but does not eliminate the possi-bility that a cyclic nucleotide may be present at a receptorsite, but is unrecognized by antibody. Studies that combinemeasurement of total nucleotide content in subcellular frac-tions with immunohistochemical localization should help todetermine if cyclic nucleotide at certain sites is not detectedby this immunohistochemical procedure.The localization of cyclic GMP in nuclear elements suggests

a role for the nucleotide in growth regulation in liver. Thesignificance of the plasma membrane staining for cyclic GMPremains to be determined. Since insulin and cholinergic agentshave been reported to raise cyclic GMP levels in liver slices

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in vitro (13), additional studies with these stimulants wouldbe of interest. The absence of significant cytoplasmic stainingfor cyclic GMP might explain the low levels (10 nM) thathave been observed for the nucleotide in this tissue.The localization of cyclic GMP on the brush border mem-

brane of the small intestinal villus suggests a role for thenucleotide in intestinal transport. It is of interest thatguanylate cyclase activity in small intestine is mostly par-ticulate (12). Further studies exploring the regulation ofguanylate cyclase and cyclic GMP action in small intestinaltransport mechanisms are indicated by the present immuno-cytochemical studies. The predominant localization of cyclicAMP in the basal and lateral sides of the cells of the villusand not at the brush border is consistent with the finding ofadenylate cyclase at these sites in rabbit intestine (11).The localization of cyclic GMP in nucleus in liver, small

intestine, adrenal cortex (14), and testis suggests that thisnucleotide serves a regulatory function in nucleus-directedevents. The most striking staining is found in nucleoli of cellsfrom these tissues, and along the chromosomes of primaryspermatocytes in testis. The role that the nucleotide serves atthese sites is not known, but this cytochemical localizationprovides clues for receptors for cyclic GMP in nuclear ele-ments. Since ribosomal RNA synthesis in nucleoli is a sitefor regulation of polymerase I (15), our studies suggest thatthe nucleotide might be involved in the control of this or othernucleolar enzymes. We have found recently that the levels ofcyclic GMP in rat adrenal increase within one hour afterhypophysectomy and decrease to below control values within15 min of adrenocorticotropic hormone administration, and inchronically hypophysectomized rats levels of cyclic GMP arenearly equal to those of cyclic AMP (14). Cyclic GMP islocated predominately in nucleoli and other nuclear elements,while cyclic AMP is found in cytoplasm of the cells of thezona fasciculata (14). When adrenal homogenates are frac-tionated by differential centrifugation in sucrose, and theabsolute amount of cyclic nucleotide is determined, we findthat nuclear elements contain approximately 50% of totalcellular cyclic GMP and less than 10% of cyclic AMP (14),confirming the immunocytochemical localization. Thesestudies suggest that cyclic GIMP may play a repressor rolein adrenal growth regulation.

In testis the demonstration of cyclic GMP on condensedchromosomes prior to meiosis, but not on chromosomes inspermatocytes at other stages of development, suggests thatthe localization of the nucleotide varies during the cell cycle.In lymphocytes (4) and fibroblasts (5) cyclic GMP levelsincrease transiently after the addition of growth-promotingfactors. While abundant evidence has accumulated whichsuggests that both cyclic AMP and cyclic GMP are involved

in the regulation of the cell cycle, the mechanisms forsuchcontrol have not been thoroughly determined. The applicationof cyclic nucleotide immunocytochemistry to cultured cellsin studies of growth regulation by growth substances shouldbe helpful in determining in greater detail the role of the cyclicnucleotides in the control of the cell cycle.The localization of both cyclic nucleotides in individual

cells in heterogeneous tissues and in specific sites within cellspoints out that the measurement of total cyclic nucleotidelevels in tissues might not be a sensitive index of cyclic nucleo-tide levels within individual cells or in cellular compartments.It is conceivable that hormonal stimulation might cause aredistribution of either cyclic nucleotide in particular tissuewithout changing the total tissue level of the nucleotide. Inpreliminary experiments, we have found that the total con-centration of cyclic GMP is unchanged in testis from 10 dayhypophysectomized rats as compared to the mature animal,and yet by immunocytochemistry membrane and particularlynuclear (not on condensed chromosomes) localization of cyclicGMP is increased. Studies in other tissues should confirmwhether this will be a general observation.

This research was supported by U.S. Public Health ServiceGrants 7R01 AM 17438-01 and AM 05330-13.

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