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
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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
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