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Vasopressin and oxytocin regulation of cyclic AMP accumulation in rat hypothalamo-neurohypophysial explants in vitro
Neuroscience Letters, I 14 (1990) 225-230
225
Elsevier Scientific Publishers Ireland Ltd. NSL 06939
Vasopressin and oxytocin regulation of cyclic AMP accumulation in rat hypothalamo-neurohypophysial explants in vitro R.B. Meeker, K.M. Michels and J.N. H a y w a r d Department of Neurology and Neurobiology Curriculum, University of North Carolina, Chapel Hill, NC (U.S.A.) (Received 1 February 1989; Revised version received 16 January 1990; Accepted 15 February 1990)
Key words." Vasopressin; Oxytocin; Acetylcholine; Cyclic AMP; Supraoptic nucleus; Pituitary; Neural lobe; Forskolin Addition of vasopressin to hypothalamo-neurohypophysial explants in vitro increased cyclic AMP accumulation whereas exogenous oxytocin decreased cyclic AMP. An opposite response pattern was observed in the neural lobe of the pituitary where vasopressin decreased and oxytocin increased cyclic AMP accumulation. Forskolin elicited a 3-fold greater increase in cyclic AMP in the neural lobe than in the supraoptic nucleus and enhanced the sensitivity of the tissues to both vasopressin and oxytocin. The ability of both vasopressin and oxytocin to modulate local cyclic AMP metabolism suggests the possibility of internal feedback within the hypothalamo-neurohypophysial system.
The neuropeptides vasopressin (VP) and oxytocin (OT) are synthesized in large quantities within magnocellular neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus for transport to the neural lobe of the pituitary and release into the general circulation. Neural control of this system is thought to be exerted by a variety of transmitters, some of which could be linked to a cyclic AMP generating system in the neurointermediate lobe (NIL). Addition of cyclic AMP or the phosphodiesterase inhibitor, theophylline, to isolated neurointermediate lobes [12] or direct activation of adenylate cyclase with forskolin [15] enhance the ability of potassium to evoke calcium-dependent VP release [1, 12, 21]. However, the receptor which might normally mediate these cyclic AMP-dependent effects is unknown. Recently, we observed that VP and OT both interacted with a cyclic AMP generating system within hypothalamo-neurohypophysial explants [13]. This interaction is consistent with the presence of a high density of VP binding sites in the neural lobe of the pituitary [2, 3, 5, 7, 22] and on the magnocellular VP and Correspondence." R.B. Meeker, Department of Neurology, CB # 7025, University of North Carolina, Chapel Hill, NC 27599, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
226 OT cell bodies in the SON and PVN [2, 7]. To explore the possibility that VP and OT might influence cyclic AMP via local receptors, we initiated a preliminary characterization of the effects of VP and OT on cyclic AMP accumulation in hypothalamoneurohypophysial explants in vitro. Hypothalamo-neurohypophysial explants were dissected from a total of 178 male, Sprague-Dawley rats (250-300 g). These explants were removed from the base of the brain within 5 min of decapitation and consisted of a 1.5-2.0 mm thick diamondshaped piece of hypothalamic tissue extending from the front of the optic chiasm to the mammillary bodies including the intact pituitary stalk and neural lobe (NL) of the pituitary with traces of the surrounding intermediate lobe [17]. Up to 6 explants were removed and immediately placed in a sterile 24-well tissue culture plate containing 2 ml of HEPES-buffered (pH 7.4) D M E M - H medium at 4°C. Vasopressin release from stimulated explants was measured by radioimmunoassay with a specific vasopressin antibody [9]. Cyclic AMP accumulation was measured utilizing a modification [19] of the method of Shimizu et al [16]. Briefly, the explants were transferred to a well containing 2 ml of HEPES-buffered D M E M - H (pH 7.4) plus 2/iCi/ml of [3H]adenine (36-40 Ci/mmol) for I h at 36°C. Uptake of [3H]adenine was found to be linear for at least 1.0 h in vitro with a rate of approximately 400 cpm" rain i. mg tissue (wet weight) for both the hypothalamus and NL. Following the [3H]adenine preincubation, the medium was aspirated and replaced with 1.9 ml of fresh medium plus 0.1 ml of drug or vehicle. After 10 min, the SON and NL were dissected from the explant, immediately placed in 1 ml of 5% trichloroacetic acid, homogenized and extracted overnight at 4°C. Chromatographic isolation of [3H]ATP and [3H]cyclic AMP was accomplished by successively passing the extract through Dowex 50X8 and alumina columns based on the method of Salomon et al. [18]. Recovery of [3H]cyclic AMP from columns run in parallel with an internal [3H]cyclic AMP standard was 60-70%. Newly synthesized cyclic AMP is expressed as the percent conversion of [3H]ATP to [3H]cyclic AMP. A series of VP, OT and acetylcholine concentrations were tested for their ability to modulate cyclic AMP. Acetylcholine was used to examine the relationship between VP release and cyclic AMP since it is thought to provide direct excitatory activation of the SON. VP and OT were added in concentrations ranging from 10 - 9 t o 10 - 5 M in order to evaluate the direct local effects of these peptides. Within each experiment the ability of each compound to affect cyclic AMP accumulation prior to and during stimulation was evaluated relative to the basal rate of accumulation (% conversion stimulated/%conversion basal). Basal accumulation of [3H]cyclic AMP in vitro was 0.96 + 0.10% conversion in the SON (n=22) and 0.80 + 0.10% conversion in the N L (n= 18). Addition of 10 -4 M acetylcholine to the explant preparation resulted in a significant increase in VP secretion from 41.3 pg/min (n= 14) to 81.6 pg/min (n= 14, P < 0.05) relative to a release of 38.7 pg/min (n = 13) for vehicle-stimulated controls. However, no change in cyclic AMP formation could be detected in the SON or N L indicating that this small amount of VP may not be sufficient to influence cyclic AMP levels. The VP- and OT-induced changes in cyclic AMP accumulation relative to basal
227
are illustrated in Fig. 1A. Vasopressin caused a small ( + 34%) but significant elevation of cyclic A M P over basal in the SON and decreased cyclic AMP content in the NL ( - 2 1 % ) at concentrations of 10 - 7 and 5 x 10 - 7 M , respectively. In contrast, OT at 10 -5 M produced exactly the opposite pattern with a 42% decrease in the SON and 30% increase in the NL. The use of forskolin with and without VP or OT provided a means of assessing the cyclic A M P generating capacity of the SON and NL as well as providing an elevated baseline for the accurate evaluation of decreases in cyclic AMP accumulation. A maximum cyclic AMP level of 5-fold over basal was observed in the NL at a forskolin concentration of 10 -5 M in comparison to a maximum stimulation of 1.6-fold over basal in the region of the SON (data not shown). Stimulation with VP and OT in the presence of 2.5 x 10 -6 M forskolin produced a similar pattern and magnitude of change in cyclic AMP as in the absence of forskolin but at VP and OT concentrations 10-1000 times lower (Fig. IB). These results provide the first evidence to suggest that VP and OT may interact with receptors in the SON and NL to modify cyclic AMP production. Changes in cyclic AMP accumulation were not apparent during small acetylcholine-induced releases of VP in vitro, but could be elicited with exogenous VP and OT at concentrations (10-8-10 -5 M) comparable to extracellular concentrations expected in the NL during moderate VP release in vivo. The NL exhibited a 3-fold greater capacity for
A
0
u3
SON
0
m
SON
NIL
02)
NIL
c3 Q_ 3; <
1.5
,•(3)
-r"
t
n
I.O -r Z 0 n~ w > Z 0
(41
0.5
(4)
VP OT 10-7M 10-SM
VP OT 5xlO-TM 10-5M
VP OT 10"SM 10-SM
. . (91
VP OT tO-SMid6M
Fig. 1. Changes in cyclic AMP accumulation in response to addition of exogenous vasopressin (VP) or oxytocin (OT) to hypothalamo-neurohypophyseal explants in vitro. Results are expressed as the change in % conversion of [3H]ATP to [3H]cyclic AMP relative to basal (A) or relative to forskolin-stimulatcd levels (B). The concentration of VP or OT used to generate each set of data is indicated below each bar and represents the lowest concentration from which significant results were observed. The number of explants run in each condition is indicated in parentheses. *P< 0.05, **P< 0.01, ***P< 0.001.
228 cyclic A M P production during forskolin stimulation than the SON, suggesting the presence of more active cyclic AMP-generating systems in the NL. Incubation of the explant with forskolin enhanced the sensitivity to both VP and OT further supporting an association with adenylate cyclase-linked cyclic AMP production. These effects of VP and OT on cyclic AMP accumulation are consistent with reports of a high density of VP and OT binding sites in the N L [2, 3, 5, 7, 22] as well as the SON and PVN [2,7]. Although small pieces of surrounding tissue may contaminate the SON and N L in our preparations, no VP or OT receptors have been observed in these overlying regions [2, 3, 5, 7]. Thus, it is likely that the target of VP and OT action are the binding sites in the SON and NL. Detailed experiments on isolated cell populations, however, will be necessary to clearly establish the cellular targets of VP and OT. The mechanism by which VP and OT exert their effects cannot be determined from the present data, but the ability of VP and OT to modulate cyclic AMP in opposite directions suggests that these neuropeptides may be working through functionally different 'receptive' mechanisms or acting in a reciprocal fashion at the same target site. The complexity of the neuropeptide action is further underscored by the differential effects of each peptide in the N L vs. the region of the SON suggesting different mechanisms of action at the level of the cell bodies vs. the terminal endings. In the NL, the possibility that presynaptic VP receptors participate in feedback control of VP and/or OT secretion is supported by the observation that VP binding sites in the N L are lost as a result of stalk transection [3]. Although the presence of presynaptic VP or OT receptors coupled to cyclic AMP metabolism is an attractive possibility, a variety of data exists which fails to find evidence for adenylate cyclase-associated VP receptors in rat brain. Tribolett et al. [20] have recently questioned the existence of specific VP and OT receptor binding in the NIL, SON and PVN. These authors found no evidence for the presence of adenylate cyclase-linked V2-receptors in any part of the rat brain. This observation is consistent with the failure of other investigators to find VP stimulation of adenylate cyclase activity in the rat brain [4, 6]. Thus, until the mechanisms of the VP- and OT-induced changes in cyclic AMP accumulation are revealed, it would be premature to conclude that such data proves the existence of adenylate cyclase-linked VP and OT receptors in the SON and NIL. For example, it is possible that VP and OT could exert their actions on cyclic AMP in an indirect manner similar to the action of VP on the anterior pituitary where VP triggers the release of PGE2 which in turn stimulates cyclic AMP formation [14]. In the anterior pituitary, the VP appears to be acting through a novel receptor designated V~b [10, I 1] which does not directly activate adenylate cyclase [8]. The mechanism of action of VP and OT in the SON and NL remains an important unanswered question. Nevertheless, our present results demonstrate: (1) that VP and OT can exert effects on cyclic AMP production in both the region of the SON and the NL; (2) these effects occur at concentrations of 10-5-10 -8 M with VP approximately 20-100 x more potent than OT; (3) the action of both peptides is facilitated in the presence of forskolin; (4) VP and OT exert opposing actions in the same region indicating that they may provide reciprocal modulation of the same target area; and (5) the SON and N L in turn respond in opposite ways to each respective peptide sug-
229
gesting that different types of feedback may be present in the region of the cell bodies and NL. These response patterns suggest complex interactions between the magnocellular VP and OT neurons and their surrounding environment and establish new possibilities for the control of neuropeptide secretion. This research was supported by NIH Javits Award NS-13411 and a Neurobiology
Training Grant. 1 Bowman, D. and Hope, D.B., Forskolin: its effects on potassium-evoked release of vasopressin from the rat neurohypophysis, Br. J. Pharmacol., 85 (1985) 197-203. 2 Brinton, R.E., Gee, K.W., Wamsley, J.K., Davis, T.P. and Yamamura, H.I., Regional distribution of putative vasopressin receptors in rat brain and pituitary by quantitative autoradiography, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 7248-7252. 3 Bunn, S.J., Hanley, M.R. and Wilkin, G.P., Autoradiographic localization of peripheral benzodiazepine, dihydroalprenolol and arginine vasopressin binding sites in the pituitaries of control, stalk transected and Brattleboro rats, Neuroendocrinology, 44 (1986) 7683. 4 Courtney, N. and Raskind, M., Vasopressin affects adenylate cyclase activity in rat brain: a possible neuromodulator, Life Sci., 32 (1983) 591-596. 5 Dashwood, M.R. and Robinson, C.A.F., Specificity of vasopressin binding to the posterior pituitary gland in the rat, Neuroendocrinology, 48 (1988) 180-187. 6 Dorsa, D.M., Majumdar, L.A., Petracca, F.M., Baskin, D.G. and Cornett, L.E., Characterization and localization of 3H-arginine-8-vasopressin binding to rat kidney and brain tissue, Peptides, 4 (1983) 699-706. 7 Freund-Mercier, M.J., Stoeckel, M.E., Dietl, M.M., Palacios, J.M. and Richard, Ph., Quantitative autoradiographic mapping of neurohypophysial hormone binding sites in the rat forebrain and pituitary gland, I. Characterization of different types of binding sites and their distribution in the LongEvans strain, Neuroscience, 26 (1988) 261-272. 8 Gaillard, R.C., Schoenenberg, P., Favrod-Coune, A., Muller, A.F., Marie, J., Bockaert, J. and Jard, S., Properties of rat anterior pituitary vasopressin receptors: relation to adenylate cyclase and the effect of corticotropin releasing factor, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 2907-2911. 9 Hayward, J.N., Murgas, K., Pavasuthipaisit, K., Perez-Lopez, F.R. and Sofroniew, M.V., Temporal patterns of vasopressin release following electrical stimulation of the amygdala and the neuroendocrine pathway in the monkey, Neuroendocrinology, 23 (1977) 61-75. 10 Jard, S., Gaillard, R.C., Guillon, G., Marie, J., Schoenenberg, P., Muller, A.F., Manning, M. and Sawyer, W.H., Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat neurohypophysis, Mol. Pharmacol., 30 (1986) 171- 177. 11 Knepel, W., Gotz, D. and Fahrenholz, F., Interaction of rat adenohypophyseal vasopressin receptors with vasopressin analogues substituted at positions 7 and 1: dissimilarity from the VI vasopressin receptor, Neuroendocrinology, 44 (1986) 390-396. 12 Mathison, R. and Lederis, K., A mechanism for adenosine 3',5'-monophosphate regulation of vasopressin secretion, Endocrinology, 106 (1980) 842-848. 13 Michels. K.M., Meeker, R.B. and Hayward, J.N., Regulation of cyclic AMP metabolism by oxytocin and vasopressin within the rat hypothalamo-neurohypophysial system, Soc. Neurosci. Abstr., 12 (1986) 449. 14 Okajima, T., Heldt, R. and Hertting, G., Functional compartmentalization of arginine-vasopressinactivated cyclic AMP in anterior pituitary gland: the presence of a compartment activated by prostaglandin E2, Life Sei., 38 (1986) 1143-1149. 15 Seamon, K.B. and Daly, J.W., Forskolin: a unique diterpine activator of cyclic AMP generating systems, J. Cycl. Nucleotide Protein Phosphorylation Res., 7 (I 981) 201-224. 16 Shimizu, H., Daly, J.W. and Creveling, C.R., A radioisotopic method for measuring the formation of adenosine 3',5'-cyclic monophosphate in incubated slices of brain, J. Neuroehem., 16 (1969) 16091619.
230 17 Sladek, C.D. and Knigge, K.M., Cholinergic stimulation of vasopressin release from the rat hypothalamo-neurohypophyseal system in organ culture, Endocrinology, 101 (1977) 411420. 18 Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylate cyclase assay, Anal. Biochem., 58 (1974) 541--548. 19 Su, Y.F., Cubbedu, L.X. and Perkins, J.P., Regulation of adenosine 3',5'-monophosphate content of human astrocytoma cells: desensitization to catecholamines and prostaglandins, J. Cycl. Nucleotide Protein Phosphorylation Res., 2 (1976) 257 270. 20 Tribolett, E., Barberis. C., Jard, S.. Dubois-Dauphin, M. and Dreifuss, J.J., Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light-microscopic autoradiography, Brain Res., 442 (1988) 105 118. 21 Vale, M.R. and Hope, D.B., Cyclic nucleotides and the release of vasopressin from the rat posterior pituitary gland, J. Neurochem., 39 (1982) 569 573. 22 Van Leeuwen, F.W. and Wolters, P., Light microscopic autoradiographic localization of[3H]arginine vasopressin binding sites in the rat brain and kidney, Neurosci. Lett., 41 (1983) 61 66.
225
Elsevier Scientific Publishers Ireland Ltd. NSL 06939
Vasopressin and oxytocin regulation of cyclic AMP accumulation in rat hypothalamo-neurohypophysial explants in vitro R.B. Meeker, K.M. Michels and J.N. H a y w a r d Department of Neurology and Neurobiology Curriculum, University of North Carolina, Chapel Hill, NC (U.S.A.) (Received 1 February 1989; Revised version received 16 January 1990; Accepted 15 February 1990)
Key words." Vasopressin; Oxytocin; Acetylcholine; Cyclic AMP; Supraoptic nucleus; Pituitary; Neural lobe; Forskolin Addition of vasopressin to hypothalamo-neurohypophysial explants in vitro increased cyclic AMP accumulation whereas exogenous oxytocin decreased cyclic AMP. An opposite response pattern was observed in the neural lobe of the pituitary where vasopressin decreased and oxytocin increased cyclic AMP accumulation. Forskolin elicited a 3-fold greater increase in cyclic AMP in the neural lobe than in the supraoptic nucleus and enhanced the sensitivity of the tissues to both vasopressin and oxytocin. The ability of both vasopressin and oxytocin to modulate local cyclic AMP metabolism suggests the possibility of internal feedback within the hypothalamo-neurohypophysial system.
The neuropeptides vasopressin (VP) and oxytocin (OT) are synthesized in large quantities within magnocellular neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus for transport to the neural lobe of the pituitary and release into the general circulation. Neural control of this system is thought to be exerted by a variety of transmitters, some of which could be linked to a cyclic AMP generating system in the neurointermediate lobe (NIL). Addition of cyclic AMP or the phosphodiesterase inhibitor, theophylline, to isolated neurointermediate lobes [12] or direct activation of adenylate cyclase with forskolin [15] enhance the ability of potassium to evoke calcium-dependent VP release [1, 12, 21]. However, the receptor which might normally mediate these cyclic AMP-dependent effects is unknown. Recently, we observed that VP and OT both interacted with a cyclic AMP generating system within hypothalamo-neurohypophysial explants [13]. This interaction is consistent with the presence of a high density of VP binding sites in the neural lobe of the pituitary [2, 3, 5, 7, 22] and on the magnocellular VP and Correspondence." R.B. Meeker, Department of Neurology, CB # 7025, University of North Carolina, Chapel Hill, NC 27599, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
226 OT cell bodies in the SON and PVN [2, 7]. To explore the possibility that VP and OT might influence cyclic AMP via local receptors, we initiated a preliminary characterization of the effects of VP and OT on cyclic AMP accumulation in hypothalamoneurohypophysial explants in vitro. Hypothalamo-neurohypophysial explants were dissected from a total of 178 male, Sprague-Dawley rats (250-300 g). These explants were removed from the base of the brain within 5 min of decapitation and consisted of a 1.5-2.0 mm thick diamondshaped piece of hypothalamic tissue extending from the front of the optic chiasm to the mammillary bodies including the intact pituitary stalk and neural lobe (NL) of the pituitary with traces of the surrounding intermediate lobe [17]. Up to 6 explants were removed and immediately placed in a sterile 24-well tissue culture plate containing 2 ml of HEPES-buffered (pH 7.4) D M E M - H medium at 4°C. Vasopressin release from stimulated explants was measured by radioimmunoassay with a specific vasopressin antibody [9]. Cyclic AMP accumulation was measured utilizing a modification [19] of the method of Shimizu et al [16]. Briefly, the explants were transferred to a well containing 2 ml of HEPES-buffered D M E M - H (pH 7.4) plus 2/iCi/ml of [3H]adenine (36-40 Ci/mmol) for I h at 36°C. Uptake of [3H]adenine was found to be linear for at least 1.0 h in vitro with a rate of approximately 400 cpm" rain i. mg tissue (wet weight) for both the hypothalamus and NL. Following the [3H]adenine preincubation, the medium was aspirated and replaced with 1.9 ml of fresh medium plus 0.1 ml of drug or vehicle. After 10 min, the SON and NL were dissected from the explant, immediately placed in 1 ml of 5% trichloroacetic acid, homogenized and extracted overnight at 4°C. Chromatographic isolation of [3H]ATP and [3H]cyclic AMP was accomplished by successively passing the extract through Dowex 50X8 and alumina columns based on the method of Salomon et al. [18]. Recovery of [3H]cyclic AMP from columns run in parallel with an internal [3H]cyclic AMP standard was 60-70%. Newly synthesized cyclic AMP is expressed as the percent conversion of [3H]ATP to [3H]cyclic AMP. A series of VP, OT and acetylcholine concentrations were tested for their ability to modulate cyclic AMP. Acetylcholine was used to examine the relationship between VP release and cyclic AMP since it is thought to provide direct excitatory activation of the SON. VP and OT were added in concentrations ranging from 10 - 9 t o 10 - 5 M in order to evaluate the direct local effects of these peptides. Within each experiment the ability of each compound to affect cyclic AMP accumulation prior to and during stimulation was evaluated relative to the basal rate of accumulation (% conversion stimulated/%conversion basal). Basal accumulation of [3H]cyclic AMP in vitro was 0.96 + 0.10% conversion in the SON (n=22) and 0.80 + 0.10% conversion in the N L (n= 18). Addition of 10 -4 M acetylcholine to the explant preparation resulted in a significant increase in VP secretion from 41.3 pg/min (n= 14) to 81.6 pg/min (n= 14, P < 0.05) relative to a release of 38.7 pg/min (n = 13) for vehicle-stimulated controls. However, no change in cyclic AMP formation could be detected in the SON or N L indicating that this small amount of VP may not be sufficient to influence cyclic AMP levels. The VP- and OT-induced changes in cyclic AMP accumulation relative to basal
227
are illustrated in Fig. 1A. Vasopressin caused a small ( + 34%) but significant elevation of cyclic A M P over basal in the SON and decreased cyclic AMP content in the NL ( - 2 1 % ) at concentrations of 10 - 7 and 5 x 10 - 7 M , respectively. In contrast, OT at 10 -5 M produced exactly the opposite pattern with a 42% decrease in the SON and 30% increase in the NL. The use of forskolin with and without VP or OT provided a means of assessing the cyclic A M P generating capacity of the SON and NL as well as providing an elevated baseline for the accurate evaluation of decreases in cyclic AMP accumulation. A maximum cyclic AMP level of 5-fold over basal was observed in the NL at a forskolin concentration of 10 -5 M in comparison to a maximum stimulation of 1.6-fold over basal in the region of the SON (data not shown). Stimulation with VP and OT in the presence of 2.5 x 10 -6 M forskolin produced a similar pattern and magnitude of change in cyclic AMP as in the absence of forskolin but at VP and OT concentrations 10-1000 times lower (Fig. IB). These results provide the first evidence to suggest that VP and OT may interact with receptors in the SON and NL to modify cyclic AMP production. Changes in cyclic AMP accumulation were not apparent during small acetylcholine-induced releases of VP in vitro, but could be elicited with exogenous VP and OT at concentrations (10-8-10 -5 M) comparable to extracellular concentrations expected in the NL during moderate VP release in vivo. The NL exhibited a 3-fold greater capacity for
A
0
u3
SON
0
m
SON
NIL
02)
NIL
c3 Q_ 3; <
1.5
,•(3)
-r"
t
n
I.O -r Z 0 n~ w > Z 0
(41
0.5
(4)
VP OT 10-7M 10-SM
VP OT 5xlO-TM 10-5M
VP OT 10"SM 10-SM
. . (91
VP OT tO-SMid6M
Fig. 1. Changes in cyclic AMP accumulation in response to addition of exogenous vasopressin (VP) or oxytocin (OT) to hypothalamo-neurohypophyseal explants in vitro. Results are expressed as the change in % conversion of [3H]ATP to [3H]cyclic AMP relative to basal (A) or relative to forskolin-stimulatcd levels (B). The concentration of VP or OT used to generate each set of data is indicated below each bar and represents the lowest concentration from which significant results were observed. The number of explants run in each condition is indicated in parentheses. *P< 0.05, **P< 0.01, ***P< 0.001.
228 cyclic A M P production during forskolin stimulation than the SON, suggesting the presence of more active cyclic AMP-generating systems in the NL. Incubation of the explant with forskolin enhanced the sensitivity to both VP and OT further supporting an association with adenylate cyclase-linked cyclic AMP production. These effects of VP and OT on cyclic AMP accumulation are consistent with reports of a high density of VP and OT binding sites in the N L [2, 3, 5, 7, 22] as well as the SON and PVN [2,7]. Although small pieces of surrounding tissue may contaminate the SON and N L in our preparations, no VP or OT receptors have been observed in these overlying regions [2, 3, 5, 7]. Thus, it is likely that the target of VP and OT action are the binding sites in the SON and NL. Detailed experiments on isolated cell populations, however, will be necessary to clearly establish the cellular targets of VP and OT. The mechanism by which VP and OT exert their effects cannot be determined from the present data, but the ability of VP and OT to modulate cyclic AMP in opposite directions suggests that these neuropeptides may be working through functionally different 'receptive' mechanisms or acting in a reciprocal fashion at the same target site. The complexity of the neuropeptide action is further underscored by the differential effects of each peptide in the N L vs. the region of the SON suggesting different mechanisms of action at the level of the cell bodies vs. the terminal endings. In the NL, the possibility that presynaptic VP receptors participate in feedback control of VP and/or OT secretion is supported by the observation that VP binding sites in the N L are lost as a result of stalk transection [3]. Although the presence of presynaptic VP or OT receptors coupled to cyclic AMP metabolism is an attractive possibility, a variety of data exists which fails to find evidence for adenylate cyclase-associated VP receptors in rat brain. Tribolett et al. [20] have recently questioned the existence of specific VP and OT receptor binding in the NIL, SON and PVN. These authors found no evidence for the presence of adenylate cyclase-linked V2-receptors in any part of the rat brain. This observation is consistent with the failure of other investigators to find VP stimulation of adenylate cyclase activity in the rat brain [4, 6]. Thus, until the mechanisms of the VP- and OT-induced changes in cyclic AMP accumulation are revealed, it would be premature to conclude that such data proves the existence of adenylate cyclase-linked VP and OT receptors in the SON and NIL. For example, it is possible that VP and OT could exert their actions on cyclic AMP in an indirect manner similar to the action of VP on the anterior pituitary where VP triggers the release of PGE2 which in turn stimulates cyclic AMP formation [14]. In the anterior pituitary, the VP appears to be acting through a novel receptor designated V~b [10, I 1] which does not directly activate adenylate cyclase [8]. The mechanism of action of VP and OT in the SON and NL remains an important unanswered question. Nevertheless, our present results demonstrate: (1) that VP and OT can exert effects on cyclic AMP production in both the region of the SON and the NL; (2) these effects occur at concentrations of 10-5-10 -8 M with VP approximately 20-100 x more potent than OT; (3) the action of both peptides is facilitated in the presence of forskolin; (4) VP and OT exert opposing actions in the same region indicating that they may provide reciprocal modulation of the same target area; and (5) the SON and N L in turn respond in opposite ways to each respective peptide sug-
229
gesting that different types of feedback may be present in the region of the cell bodies and NL. These response patterns suggest complex interactions between the magnocellular VP and OT neurons and their surrounding environment and establish new possibilities for the control of neuropeptide secretion. This research was supported by NIH Javits Award NS-13411 and a Neurobiology
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