Proc. Natl. Acad. Sci. USA Vol. 86, pp. 5198-5201, July 1989 Neurobiology Cholecystokinin evokes secretion of oxytocin and vasopressin from rat neural lobe independent of external calcium (neurohypophysis/receptors/neurosecretion/neuropeptide/protein kinase C) C. A. BONDY*t, R. T. JENSEN*, L. S. BRADY§, AND H. GAINER* *Laboratory of Neurochemistry, National Institute of Neurological and Communicative Disorders and Stroke, tDigestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and §Unit on Functional Neuroanatomy, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 Communicated by William A. Hagins, April 5, 1989 ABSTRACT Cholecystokinin (CCK) and its receptors are albumin (1.5 mg/ml)] maintained at 370C and equilibrated abundantly represented in the central nervous system. However, with frequent washes for 90 min prior to starting the exper- a specific role or mechanism ofaction for CCKin this context has iment. The incubation and stimulus assembly have been not been established. CCK coexists with oxytocin in magnocel- described in detail elsewhere (4). The stream of 02 suffusing lular neurons of the hypothalamic-neurohypophysial system, the medium was turned offduring periods of peptide addition sharing common neurosecretory vesicles with oxytocin in the (and for the same period in control preparations) to avoid neural lobe of the pituitary. The neural lobe, which consists oxidation of CCK and related peptides, which can destroy primarily of oxytocin- and vasopressin-containing axons and their activity. Medium was collected at 5- or 10-min intervals nerve terminals and their surrounding glia, provides a relatively for determination of OT and VP content by specific radio- simple model system allowing for the study of the regulation of immunoassay as described (4). Peptides werf obtained from neurosecretion at the nerve terminal level, free from the complex Peninsula Laboratories, the CCK antagonist L-364,718 was array of synaptic effects present throughout the rest of the from Merck Sharp & Dohme, and staurosporine was from central nervous system. In this paper, we demonstrate the Kyowa Medex (Tokyo). presence ofhigh-affinity CCK binding sites in the rat neural lobe For CCK receptor studies, pituitaries were dissected from and show that activation of these receptors by the sulfated male Sprague-Dawley rats, rinsed in room temperature nor- octapeptide, CCK-8, and related peptides causes potent secre- mal saline, and frozen directly in Lab-Tek O.C.T. compound tion of oxytocin and vasopressin from the isolated nerve termi- embedding matrix over dry ice. They were cut into 12- nals. The secretagogue action of CCK-8, which is blocked by a ,m-thick sections at -15'C and thaw mounted onto gelati- CCK receptor antagonist (L-364,718), is independent of electri- nized slides. After drying, they were preincubated in 50 mM cal stimulation and extracellular calcium and is blocked by an Tris-HCl (pH 7.4) containing 0.5% bovine serum albumin for inhibitor of protein kinase C. Thus, the action of CCK on the 20 min at 240C, then incubated in 10 mM Hepes (pH 6.5) neural lobe provides an example of peptide ligand-induced containing 0.5% bovine serum albumin, 0.025% bacitracin, neurosecretion apparently mediated by second messengers leupeptin (4 gg/ml), 130 mM NaCl, 4.5 mM KCI, 5 mM rather than depolarization-induced calcium influx. MgCl2, 1 mM EGTA, and 0.125 nM 1251I-labeled CCK-8 (Amersham) for 2 hr at 240C. After the incubation, slides Since cholecystokinin (CCK) is colocalized in neurosecre- underwent six 15-min washes in 5% bovine serum albumin at tory granules with oxytocin (OT) in the hypothalamic- 40C and were dried and apposed to LKB Ultrofilm for 12 neurohypophysial system (18), it would be expected that days. these two peptides are co-released from the nerve terminals of the neural lobe (NL). Supporting this view are the obser- vations that CCK immunoreactivity is lost as a result of stalk RESULTS section, and physiologic stimuli that result in depletion ofOT To test the effect of CCK on electrically evoked secretion also cause a marked depletion of NL CCK (1). The molar from the NL, we used a low-frequency, low-intensity elec- concentration of CCK in the rat NL is several orders of trical stimulus protocol that elicits relatively low but reliable magnitude less than that of OT (1), suggesting that the CCK levels of OT and VP secretion. This protocol was chosen to secreted with OT plays a local rather than systemic role. minimize the release of endogenous copeptides, so as not to Since other colocalized peptides-namely, dynorphin (2) and obscure their effects when added experimentally (2). It was corticotropin-releasing factor (3)-have been found to play a found that CCK-8 (0.1 nM) did not affect the electrically role in the regulation of neurohypophysial hormone secre- evoked release of OT (Fig. 1A) or VP (unpublished data) but tion, we investigated the effects of CCK on the release of OT instead caused an episode of secretion that proved to be and vasopressin (VP) from isolated rat NLs. completely independent of electrical stimulation (Fig. 1B). Furthermore, unlike the immediate response to electrically MATERIALS AND METHODS stimulated secretion, CCK-evoked secretion was delayed, reaching a peak 40-50 min after first exposure to the peptide Pituitaries were removed from male Sprague-Dawley rats (Fig. 1). (200-250 g) and NLs were freed from intermediate lobe tissue No prior episode of electrical stimulation (i.e., S1) was under a dissecting microscope. NLs were singly impaled on in stimulating electrodes immersed in oxygenated physiologic necessary for the action ofCCK, as was found experiments saline [140 mM NaCl/4.5 mM KCl/1.5 mM MgCl2/2.0 mM in which electrical stimulation was omitted altogether (un- CaCl2/10 mM Hepes, pH 7.3/10 mM dextrose/bovine serum Abbreviations: CCK, cholecystokinin; CCK-8, cholecystokinin sul- fated octapeptide; NL, neural lobe; OT, oxytocin; VP, vasopressin; The publication costs of this article were defrayed in part by page charge PKC, protein kinase C. payment. This article must therefore be hereby marked "advertisement" tTo whom reprint requests should be addressed at: Building 36, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Room 4D20, National Institutes of Health, Bethesda, MD 20892. 5198 Downloaded by guest on September 25, 2021 Neurobiology: Bondy et al. Proc. Natl. Acad. Sci. USA 86 (1989) 5199 A 0 OCK * CONTROL ba 0 C/) 2 80 Minutes o 10o12 10-11 1010 10-9 10-8 10o7 B 2.5 - CCK-8 logM FIG. 2. Dose-response relationship for stimulation by CCK-8 of 2.0 - VP and OT release from isolated rat NLs. Experiments were performed as shown in Fig. 1B. Data are expressed as S2/S1 ratios, 1.5 - which were determined for each individual preparation as follows: 04 hormone released over 30 min following the standard electrical to stimulus (subtracting basal release determined in the period imme- 1.0 - diately preceding the stimulus) = S1; hormone released during the 110 min following exposure to test substances (subtracting basal level 0.5 n of hormone secretion demonstrated in the period prior to exposure) = S2. Each point represents the mean ± SEM of five to seven n% 1 _ experiments at each concentration. The zero point represents S2/S1 40 80 120 160 200 values for controls (no peptide exposure during the S2 period). r_ Minutes exposure to CCK or related peptides, as explained in the 0 C calcium-free legend to Fig. 2. 1.5 - Electrically evoked secretion from the nerve terminals of * the NL is dependent on depolarization-induced calcium influx (5) and does not occur when calcium is absent from the external medium, as shown in Fig. 1C, where release to the 1.0- second period of electrical stimulation, indicated by the arrow, is abolished. CCK-8, however, evoked OT and VP 04 secretion in calcium-free medium, as shown in Fig. 1C and 0.5- also in Table 1 (in the latter experiments, there was no second electrical stimulation). CCK also produced secretion in me- Table 1. Effects of CCK-8 and related peptides on VP and OT secretion from isolated rat neural lobes 40 80 1 20 Minutes S2/S1 Treatment OT VP FIG. 1. CCK-8 stimulates secretion from isolated NLs indepen- ± ± dently of electrical stimulation. (A) Effects of CCK-8 (0.1 nM) on CCK-8 (1 nM) 6.689 1.032 4.157 0.636 electrically evoked secretion of OT using a low-frequency double CCK-8 (1 nM), calcium-free 6.054 ± 0.641 4.202 ± 0.545 electrical stimulus (ES) protocol. In these experiments, each NL CCK-8 (1 nM) + staurosporine 0.777 ± 0.246* 0.935 ± 0.344* received two identical periods of ES (arrows), consisting of 1000 CCK-8 (1 nM) + L-364,718 5.874 ± 0.854 3.567 ± 0.873 pulses of 3 V and 0.5 ms at 6 Hz. Controls (o; n = 3) underwent both CCK-8 (0.1 nM) 3.330 ± 0.532 2.549 ± 0.226 stimuli in control medium, while experimental animals (o; n = 3) CCK-8 (0.1 nM), nonsulfated 2.810 ± 0.700 3.432 ± 0.817 were exposed to CCK during the second stimulus period, as shown CCK-4 (0.1 nM) 0.278 ± 0.094* 0.493 ± 0.233t by the solid bar along the abscissa. (B) Experiments (n = 6) in which Gastrin I (0.1 nM) (human) 2.535 ± 0.297 3.162 ± 0.969 the second ES was omitted and the effect ofCCK (0.1 nM) on resting CCK-8 (0.1 nM) + L-364,718 0.332 ± 0.067* 0.159 ± 0.091* secretion is shown.