Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3510-3512, May 1986 Neurobiology Role of cholecystokinin in corticotropin release: Coexistence with vasopressin and corticotropin-releasing factor in cells of the rat hypothalamic paraventricular nucleus (neuroendocrinology/neuropeptides/corticotropin regulation) EVA MEZEY*, TERRY D. REISINE*, LANA SKIRBOLLt, MARGERY BEINFELDt, AND J6ZSEF Z. KIss*§ *Laboratory of Cell Biology and tClinical Neuroscience Branch, National Institute of Mental Health, Bethesda, MD 20892; and tDepartment of Pharmacology, St. Louis University, School of Medicine, St. Louis, MO 63104 Communicated by J. Szentdgothai, December 18, 1985 ABSTRACT Cholecystokinin-8 (CCK8)-containing cell gelatin-coated slides. For details of the immunofluorescent bodies in the parvocellular region of the rat paraventricular staining, see Hokfelt et al. (15). Briefly, the brains were nucleus (PVN) contain vasopressin and corticotropin-releasing postfixed for 90 min, washed overnight, and sectioned in a factor (CRF). The CCK8 and vasopressin in these cells can cryostat. The 5-,um-thick sections were incubated with pri- readily be visualized in adrenalectomized, but not in sham- mary antibodies [a mixture of a rabbit polyclonal anti-CCK operated animals. Furthermore, CCK8 levels as measured by antiserum and a mouse monoclonal anti-vasopressin-neuro- RIA change in the PVN and in the median eminence in response physin (VP-NP) antibody, both at final dilution of 1:500] in to adrenalectomy. CCK8 has a stimulatory effect on cortico- 10% normal goat serum, 0.6% Triton X-100, and PBS. The tropin (ACTH) release from primary cultures of the anterior sections were incubated for 2 hr at room temperature fol- pituitary. This stimulation is additive with that produced by lowed by an overnight incubation at 4°C. Then they were vasopressin; CCK8 plus vasopressin have an effect as great as rinsed and incubated in a phosphate-buffered solution (pH CRF in stimulating ACTH release. Our results suggest that 7.4, 0.1 M) containing rhodamine-conjugated anti-rabbit and CCK8 may participate in the regulation ofACTH release under fluorescein-conjugated anti-mouse secondary antibodies certain physiological conditions. (1:100) for 1 hr at room temperature. The sections were mounted with glycerol diluted in PBS (3:1) and viewed with Cholecystokinin (CCK), a gastrointestinal hormone is pres- a Leitz fluorescent microscope. After photographs were ent in the brain (1-3). It has been found in the hypothala- taken, the immunostaining was eluted with acid potassium mo-hypophyseal system, in neurons of the paraventricular permanganate according to Tramu et al. (16). The sections (PVN) and supraoptic nuclei which also contain oxytocin were reincubated with the rhodamine-conjugated secondary (4-7). Both the internal and the external layers ofthe median antibody and examined to be sure that there was no residual eminence (ME) have CCK8 immunoreactive fibers (8). A staining. Finally, they were incubated with the third primary population of parvocellular neurons in the PVN that contain antibody (CRF) and processed as described above. Photo- CCK8 and project to the external layer has recently been graphs were taken and compared to the others. The CRF described (9). Thus, while the "magnocellular" CCK system antiserum used was raised in rabbit against rat CRF and its is thought to be composed of neurosecretory neurons that immunocytochemical specificity was described earlier (17). project to the posterior pituitary and release CCK plus The CCK antibody was raised against CCK8 sulfate in oxytocin (4-7), the "parvocellular" CCK8 system may play rabbits; the preparation and specificities of the antiserum a role in regulating the function of the anterior pituitary. In have been reported (9, 18). The VP-NP was stained with a fact, it has been suggested that CCK8 may regulate the monoclonal antibody (PS 41) that has been reported to be activity of corticotrophs, but its precise role is still contro- highly specific (19). CCK8 (20 ,ug per ml ofantibody solution) versial (8, 10-14). Our present study was based on the blocked staining by the anti-CCK antibody; likewise, VP-NP following observations: (i) The distribution of the (20 ,ug/ml) and CRF (20 ,ug/ml) blocked staining by the "parvocellular" CCK8 cell bodies was similar to that of VP-NP and CRF antibodies, respectively. Absorbing with corticotropin-releasing factor (CRF) immunopositive neu- CCK did not affect the VP-NP or CRF staining; nor did VP, rons in the PVN (9). (ii) The CCK present in the external zone vasoactive intestinal polypeptide (VIP), or PHI at concen- of the ME strongly responds to changes in the adrenal-pitu- trations of 25 ,ug/ml. Absorption of the CCK antibody with itary axis (8). CRF also did not affect the staining. Tissue Culture. Primary pituitary cultures were prepared from male Sprague-Dawley rats by removing the pituitary MATERIALS AND METHODS separating the anterior lobe and placing them in PBS (20). The Male Sprague-Dawley rats (200 g) were used in all studies. cells were enzymatically dispersed with collagenase (10 Immunostaining Procedures. The animals were perfused units/ml), DNase (1 mg/ml), and 0.2% trypsin in the presence with 4% paraformaldehyde containing 0.2% picric acid in 0.1 of 1% antibiotics. The cells were placed in Dulbecco's M sodium phosphate buffer (pH 7.4). Five rats received modified Eagle's medium (1 ml) in 10% fetal calf serum, intraventricular injection of colchicine (120 ,ug) 2 days prior streptomycin (0.5%), PenG (0.5%), and 1% nonessential to perfusion. The brains were removed and postfixed in the amino acids. Cells were plated at a density of200,000 per well same fixative for 1 hr. After washing overnight in phosphate- and kept in a humidified incubator at 37°C in an atmosphere buffered saline (PBS) containing 5% sucrose at 4°C, 5-,um- of 10% for 3 days. The medium in which the cells were grown thick frozen sections were cut in a cryostat and thawed on Abbreviations: CCK, cholecystokinin; PVN, parvocellular nucleus; CRF, corticotropin-releasing factor; ACTH, corticotropin; ME, The publication costs of this article were defrayed in part by page charge median eminence; VP, vasopressin; NP, neurophysin. payment. This article must therefore be hereby marked "advertisement" §Present address: Institute of Experimental Medicine, Hungarian in accordance with 18 U.S.C. §1734 solely to indicate this fact. Academy of Sciences, Budapest, H-1450 Hungary. 3510 Neurobiology: Mezey et al. Proc. Natl. Acad. Sci. USA 83 (1986) 3511 brains were sliced fresh under a stereomicroscope and the regions were removed by the Palkovits' "punch" technique (22). The tissue samples were placed in ice-cold 0.1 M HC1 and sonicated. The homogenates were neutralized with 0.1 M NaOH and assayed directly for CCK8 immunoreactivity by a RIA as described (23). The homogenates were kept ice-cold during the whole procedure. RESULTS In addition to the first described magnocellular CCK cells, our immunocytochemical studies also revealed a large pop- ulation ofparvocellular CCK8 immunoreactive neurons in the PVN. This group of cells could only be visualized in adre- nalectomized rats that received colchicine (120 jig) intraven- tricularly 2 days prior to being killed. Examination of adja- cent serial sections (data not shown), as well as restaining of the same sections, revealed that many of these CCK8 immunopositive cell bodies also contain VP-associated NP and CRF (Fig. 1). To study the functional significance ofthis coexistence, we measured ACTH release from primary cultures of anterior pituitary and determined brain CCK8, levels after adrenal- ectomy. Our in vitro studies showed a dose-dependent stimulatory effect of CCK8 on ACTH release; CCK8 caused increases of 41%, 47%, and 147% in ACTH secretion over basal levels at concentrations of 10-11, 10-9, and 10-7 M, respectively (Table 1). CCK8 did not seem to affect release of ACTH induced by CRF, which by itself produces as great a release of ACTH as we could observe. However, CCK8 and VP together were able to release almost as much ACTH as a maximally effective dose of CRF (Table 1). Adrenalectomy did not appear to affect CCK8 turnover in magnocellular neurons; there was no change in CCK8 immunoreactivity in the supraoptic nucleus or posterior pituitary of adrenalectomized rats (Table 2). On the other hand, adrenalectomy caused a marked decrease in CCK8 levels in the ME and a modest, but significant, reduction in the PVN. DISCUSSION The above results show that CCK8 coexists with CRF in cells of the parvocellular subdivision of the PVN. Earlier studies have indicated that these same cells also produce VP in response to adrenalectomy (24-26). Our data show that CCK8, VP-NP and CRF coexist in some PVN cells. Since VP-NP and VP itself are always colocalized in the same neurosecretory vesicles (27), the VP-NP immunopositivity indicates the presence of VP in the cells. Corticotropin-releasing hormone (28) is the most potent FIG. 1. Immunostaining of CCK-, VP-NP-, and CRF-containing stimulant of ACTH secretion known, but VP stimulates neurons in the hypothalamic paraventricular nucleus. VP-NP is a ACTH secretion as well. Similarly, CCK8, the third peptide specific marker for VP-containing cells. The same section is shown found in "CRF" neurons has also been shown to stimulate stained consecutively with antibodies against CCK, VP-NP, and M CRF. Arrows point to neurons that are positive for all three peptides. ACTH release from pituitary quarters (at a 10-7 concen- Many neurons lacking one or more of the peptides can also be observed. Table 1. Effect of CCK8 on ACTH release from anterior pituitary cells was removed and fresh medium containing 25 mM Hepes (pH CCK8 CCK8 CCK8 7.4) was added. Cells were incubated with or without CRF, arginine vasopressin (AVP), CCK8 (Bachem Fine Chemicals, Condition Basal (10 pM) (1 ,uM) (0.1 ,uM) Torrance, CA), or a combination of the three for 3 hr.