Dec 18, 1985 - oxytocin (4-7), the "parvocellular" CCK8 system may play a role in regulating ..... Ivy, A. C. & Oldberg, E. (1928) Am. J. Physiol. 85, 381-383. 4.
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 bodies in the parvocellular region of the rat paraventricular nucleus (PVN) contain vasopressin and corticotropin-releasing factor (CRF). The CCK8 and vasopressin in these cells can readily be visualized in adrenalectomized, but not in shamoperated animals. Furthermore, CCK8 levels as measured by RIA change in the PVN and in the median eminence in response to adrenalectomy. CCK8 has a stimulatory effect on corticotropin (ACTH) release from primary cultures of the anterior pituitary. This stimulation is additive with that produced by vasopressin; CCK8 plus vasopressin have an effect as great as CRF in stimulating ACTH release. Our results suggest that CCK8 may participate in the regulation of ACTH release under certain physiological conditions.
gelatin-coated slides. For details of the immunofluorescent staining, see Hokfelt et al. (15). Briefly, the brains were postfixed for 90 min, washed overnight, and sectioned in a cryostat. The 5-,um-thick sections were incubated with primary antibodies [a mixture of a rabbit polyclonal anti-CCK antiserum and a mouse monoclonal anti-vasopressin-neurophysin (VP-NP) antibody, both at final dilution of 1:500] in 10% normal goat serum, 0.6% Triton X-100, and PBS. The sections were incubated for 2 hr at room temperature followed by an overnight incubation at 4°C. Then they were rinsed and incubated in a phosphate-buffered solution (pH 7.4, 0.1 M) containing rhodamine-conjugated anti-rabbit and fluorescein-conjugated anti-mouse secondary antibodies (1:100) for 1 hr at room temperature. The sections were mounted with glycerol diluted in PBS (3:1) and viewed with a Leitz fluorescent microscope. After photographs were taken, the immunostaining was eluted with acid potassium permanganate according to Tramu et al. (16). The sections were reincubated with the rhodamine-conjugated secondary antibody and examined to be sure that there was no residual staining. Finally, they were incubated with the third primary antibody (CRF) and processed as described above. Photographs were taken and compared to the others. The CRF antiserum used was raised in rabbit against rat CRF and its immunocytochemical specificity was described earlier (17). The CCK antibody was raised against CCK8 sulfate in rabbits; the preparation and specificities of the antiserum have been reported (9, 18). The VP-NP was stained with a monoclonal antibody (PS 41) that has been reported to be highly specific (19). CCK8 (20 ,ug per ml of antibody solution) blocked staining by the anti-CCK antibody; likewise, VP-NP (20 ,ug/ml) and CRF (20 ,ug/ml) blocked staining by the VP-NP and CRF antibodies, respectively. Absorbing with CCK did not affect the VP-NP or CRF staining; nor did VP, vasoactive intestinal polypeptide (VIP), or PHI at concentrations of 25 ,ug/ml. Absorption of the CCK antibody with CRF also did not affect the staining. Tissue Culture. Primary pituitary cultures were prepared from male Sprague-Dawley rats by removing the pituitary separating the anterior lobe and placing them in PBS (20). The cells were enzymatically dispersed with collagenase (10 units/ml), DNase (1 mg/ml), and 0.2% trypsin in the presence of 1% antibiotics. The cells were placed in Dulbecco's modified Eagle's medium (1 ml) in 10% fetal calf serum, streptomycin (0.5%), PenG (0.5%), and 1% nonessential amino acids. Cells were plated at a density of 200,000 per well and kept in a humidified incubator at 37°C in an atmosphere of 10% for 3 days. The medium in which the cells were grown
Cholecystokinin (CCK), a gastrointestinal hormone is present in the brain (1-3). It has been found in the hypothalamo-hypophyseal system, in neurons of the paraventricular (PVN) and supraoptic nuclei which also contain oxytocin (4-7). Both the internal and the external layers of the median eminence (ME) have CCK8 immunoreactive fibers (8). A population of parvocellular neurons in the PVN that contain CCK8 and project to the external layer has recently been described (9). Thus, while the "magnocellular" CCK system is thought to be composed of neurosecretory neurons that project to the posterior pituitary and release CCK plus oxytocin (4-7), the "parvocellular" CCK8 system may play a role in regulating the function of the anterior pituitary. In fact, it has been suggested that CCK8 may regulate the activity of corticotrophs, but its precise role is still controversial (8, 10-14). Our present study was based on the following observations: (i) The distribution of the "parvocellular" CCK8 cell bodies was similar to that of corticotropin-releasing factor (CRF) immunopositive neurons in the PVN (9). (ii) The CCK present in the external zone of the ME strongly responds to changes in the adrenal-pitu-
itary axis (8).
MATERIALS AND METHODS Male Sprague-Dawley rats (200 g) were used in all studies. Immunostaining Procedures. The animals were perfused with 4% paraformaldehyde containing 0.2% picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Five rats received intraventricular injection of colchicine (120 ,ug) 2 days prior to perfusion. The brains were removed and postfixed in the same fixative for 1 hr. After washing overnight in phosphatebuffered saline (PBS) containing 5% sucrose at 4°C, 5-,umthick frozen sections were cut in a cryostat and thawed on
Abbreviations: CCK, cholecystokinin; PVN, parvocellular nucleus; CRF, corticotropin-releasing factor; ACTH, corticotropin; ME, median eminence; VP, vasopressin; NP, neurophysin. §Present address: Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, H-1450 Hungary.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3510
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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 population of parvocellular CCK8 immunoreactive neurons in the PVN. This group of cells could only be visualized in adrenalectomized rats that received colchicine (120 jig) intraventricularly 2 days prior to being killed. Examination of adjacent 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 of this coexistence, we measured ACTH release from primary cultures of anterior pituitary and determined brain CCK8, levels after adrenalectomy. 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
FIG. 1. Immunostaining of CCK-, VP-NP-, and CRF-containing in the hypothalamic paraventricular nucleus. VP-NP is a specific marker for VP-containing cells. The same section is shown stained consecutively with antibodies against CCK, VP-NP, and CRF. Arrows point to neurons that are positive for all three peptides. Many neurons lacking one or more of the peptides can also be observed. neurons
was removed and fresh medium containing 25 mM Hepes (pH 7.4) was added. Cells were incubated with or without CRF, arginine vasopressin (AVP), CCK8 (Bachem Fine Chemicals, Torrance, CA), or a combination of the three for 3 hr. Medium was removed and corticotropin (ACTH) immunoreactivity was measured as described (21). Values are the mean SEM of triplicate experiments. This experiment was repeated twice with similar results. The CCK8 used in these studies was verified by HPLC. Biochemistry. Sprague-Dawley male rats were decapitated 8 days after bilateral adrenalectomy or sham operations. The ±
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 stimulant of ACTH secretion known, but VP stimulates ACTH secretion as well. Similarly, CCK8, the third peptide found in "CRF" neurons has also been shown to stimulate ACTH release from pituitary quarters (at a 10-7 M concenTable 1. Effect of CCK8 on ACTH release from anterior pituitary cells CCK8 CCK8 CCK8 (1 ,uM) (0.1 ,uM) Basal (10 pM) Condition Control 1.7 ± 0.24 2.4 ± 0.3 2.6 ± 0.2* 4.3 ± 0.35t CRF (0.1 ,uM) 12.1 ± 1.1t 11.9 + 0.4* 12.0 ± 1.3t 13.3 ± 0.8t AVP 4.3 ± 0.6t 4.0 0.5t 5.8 ± 0.4* 10.8 ± 1.2* (1 ,uM) Results are expressed as ng of immunoreactive ACTH per well. AVP, arginine vasopressin. *p < 0.05; tP < 0.01; tP < 0.001 different than control basal levels.
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Table 2. Effect of adrenalectomy on CCK8-like immunoreactivity (ng per mg of protein) in specific brain areas and the posterior pituitary Supraoptic Posterior ME PVN nucleus pituitary Adrenalectomized 1.95 ± 0.37* (16) 2.19 ± 0.39t (16) 1.48 ± 0.09 (8) 1.54 ± 0.16 (8) Control 5.42 ± 0.69 (16) 4.08 ± 0.46 (15) 1.62 ± 0.19 (8) 1.30 ± 0.11 (7) Numbers of animals are given in parentheses. *P < 0.001; tP < 0.01.
tration) (14). The authors' conclusion in this previous study was that the concentration of CCK8 is rather unlikely to be found in portal blood under physiological conditions. Using primary cultures of anterior pituitary cells, we have demonstrated that a lower concentration (10-9 M) of CCK8 has a significant effect on ACTH release, suggesting that the CCK8 in the ME may have a direct effect on the anterior pituitary and that it may participate in regulating ACTH release. Certain conditions-such as adrenalectomy-seem to concomitantly change the levels of CCK8 and VP in the PVN neurons. The in vitro results suggest that these two peptides together have a stimulatory effect on ACTH release comparable to that of CRF. Under circumstances in which the ACTH-releasing abilities of CRF are diminished, such as during CRF-receptor desensitization (20), CCK8 and VP may compensate for this loss of effect. The decrease in CCK8 levels in the PVN but not in the supraoptic nucleus suggests that adrenalectomy may affect CCK metabolism in the parvocellular vs. the magnocellular neuronal population. The marked depletion of CCK8 from the ME probably reflects a substantial increase in its release (CRF levels in the ME also drop after adrenalectomy) (ref. 28; F. Antoni, personal communication). This is analogous to the depletion of VP from the posterior pituitary seen when animals are deprived of water. That there is an increase in CCK immunostaining in the PVN of colchicine-treated adrenalectomized rats seems paradoxical at first glance, but colchicine blocks transport and release of neurotransmitters. It seems likely that adrenalectomy provokes an increase in CCK release as well as a compensatory increase in its biosynthesis. When axonal transport and release are inhibited by colchicine, this compensatory increase is reflected in the enhanced perikaryal staining observed. The circumstances under which CRF, VP, and/or CCK8 are released are not known. The peripheral and central factors that regulate the synthesis and release of these peptides remain to be investigated. We thank Dr. H. Gainer for the VP-NP antibody, Dr. W. Vale for the CRF antiserum, Patricia Thurston and Melitta Kiss for their secretarial help, and Larry Ostby for the photographic work. The work of M.B. was supported in part by National Institutes of Health Grants NS 18335 and NS 18667. (London)
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