J. Endocrinol. Invest. 1 : 215, 1978
Transmitter mediated arginine vasopressin release from supedused hypothalamus and pituitary gland 1 H. Wagner, H. Haberle,
v.
Maier*, and R.E. Lang*
Sektion Nephrologie und 'Department fUr Endokrinologie und Stoffwechsel des Zentrum fUr Innere Medizin und Kinderheilkunde der Universitiit Ulm, Ulm, GFR
ABSTRACT. The study was designed to investigate the effect of various neurotransmitters on the hypothalamus and pituitary gland to determine the sites of their action. Superfused isolated rat hypothalami and pituitary glands demonstrated basal secretion of arginine vasopressin (AVP) and repeated response to stimulation thus showing the viability of the preparation. Acetylcholine and histamine stimulated the release of AVP at the hypothalamic and pituitary levels; dopamine and norepinephrine released AVP in a dose related manner only from the hypothalamus;· angiotensin 11 released AVP in the same fashion only from the pituitary gland. AVP secretion stimulated by dopamine and norepinephrine may represent synaptic inputs which are localized at the hypothalamus and must be distinguish~d from the site of action at the pituitary gland of angiotensin 11. hypothalamus was cut off in a block, using iris scissors. The tissue block was limited rostrally by the anterior border of the optic chiasma, laterally by the hypothalamic fissures, and caudally by the mammillary bodies. The dorsal extent of the cut was to a depth of 2 mm. The explant was gently removed at the cut ophtalmic stalk and was transferred immediately to a plastic vial containing the incubation fluid. Thereafter the entire pituitary gland was gently removed from the sella and was also transferred to a second vial. The medium used for incubation and superfusion resembled cerebrospinal fluid and contained (in mM): NaCI, 126; KCL, 6; NazHP04, 1; MgS04. 7 H20, 0.88; NaHC03, 22; CaCI2, 1.45; O-glucose 0.2%, gassed continously with carbogen (02/C02 = 9515 wtlvol) to pH 7.4, osmolality 298 mosmol. The incubation period lasted 20 min. The tissue, used either as a block or sliced in two halves, was thereafter transferred to superfusion chambers (Millipore no. 10SX0001300). Each plastic chamber, containing three organs in a volume of 0.85 ml, was kept at 37 C in a water bath. Two experiments were run in parallel. A constant flow rate of 0.8 ml/min was maintained by a peristaltic pump (Oesaga, Heidelberg) and, the samples were collected in plastic vials at 2 min intervals. After a 20 min equilibration period, the test substance was added by a sidearm. The stimulation period lasted 10 min. Thereafter a buffer period followed and a further stimulation period was set up. The superfusion period lasted 100 minutes. All collected samples were assayed immediately after the experiment. The AVP content of homogenized hypothalami and pituitary glands was determined after extraction using 0.1 N HCI ethanol. The extract was blown to dryness and assayed; stimulated AVP release was expressed
INTRODUCTION The magnocellular secretory system has been shown to form neurosecretory material in the perikaria of the supraoptic and paraventricular neurons which is transported to the pituitary gland in their axons (1, 29). Immunochemical studies have demonstrated the presence of aminergic and cholinergic fibers in the hypothalamus as well as in the anterior and posterior pituitary gland lobes of the (18, 21). This may lead to the assumption that the secretion of the neurohormones, arginine vasopressin (AVP) and oxytocin, is not only controlled by synaptic inputs at the hypothalamic but also at the hypophysial level. In vitro experiments using the isolated neural lobe (12), the entire hypothalamo-neurohypophysial system (15, 24) and isolated stJpraoptic cells (27) have shown that adrenergic and cholinergic neurons are involved in the AVP release. The purpose of the present study was to investigate the control of AVP release by different putative neurotransmitters at the hypothalamic and 1or the neurohypophysial level. The model of superfused pituitary glands and hypothalami was used to study the dynamics of hormone release.
MATERIALS AND METHODS Male Sprague Oawley rats (120-150 g) were decapitated, the skull was opened and the brain reflected. The
1 Presented
In
part at the XI th Acta Endocrinologica Congress Lausanne, 1977
(37) and part of the thesis of Haberle
Key-words· Arginine vasopressin release, hypophysIs, hypo1halamus. neurotransmitters. superfuslon Correspondence- Or. Hans Wagner, Steinhbvelstr. 9. 0-79 Ulm/Oonau Received September 24. 1977: accepted May 2. 1978
215
H. Wagner, M. Haber/e, V. Maier, and RE Lang
as percent of the basal secretion. Baseline AVP release was determined between the 20th and 40th min. The hypothalami and pituitary glands were examined, in some experiments, by light microscopy for completeness and integrity before and after superfusion.
12.0 ± 1.8 j.lU/organ/1 0 min. The AVP release from a whole compared with two halves of a hypothalamus and pituitary gland was similiar. The AVP extracted from organs, before as well as after the superfusion, amounted to 12.8 mU per hypothalamus and 320 mU per pituitary gland. This shows that the basal release of AVP from the hypothalamic area over 10 minutes is about twenty times higher than that of the pituitary (Table 1) when expressed as % of total hormone content, indicating that the hypothalamus has more hormone in a "releasable form".
Arginine vasopressin radioimmunoassay
200 j.l1 antibody (final dilution 1 : 20,000) was incubated with 100 j.l1 ' 25 1 arginine vasopressin (5000 cpm) and 100 j.l1 standard or superfusion fluid for 24 h; thereafter 50 j.l1 second antibody (anti-rabbit-globuline forte, Behring Frankfurt) was added and after a second 24 h period the incubate was separated by centrifugation (2000 g) for 10 min (36) . Drugs used in the studies were obtained commercially: norepinephrine, dopamine, acetylcholine and nicotine (Fluka AG - Buchs, Switzerland); angiotensin and phentolamine (Ciba Geigy, Wehr). Synthetic arginine vasopressin was a gift from Or. J. Mulder, (Ferring AB Malmo, Sweden).
Hypotha/amus
Stimulations of superfused hypothalami were performed, after a preincubation time of 20 minutes and a further equilibration period of 12 - 20 minutes, with acetylcholine (ACH), norepinephrine (NA), dopamine (DA), histamine (HA), serotonin (OHTA) and angiotensin II (A 11). The time of exposure lasted 10 minutes and during this period the maximum of the AVP release was obtained after 2 - 4 minutes (Fig. 1). During prolonged exposure to the drugs the secretion returned to basal values after 10 - 14 minutes, while stimulation was continued (Fig. 3). After a control period of 60 minutes a repeated stimulation yielded identical peaks over a 120 min. superfusion period (Fig. 2). Significance of the results was tested by the Student's t test and significance was defined as p
Transmitter mediated arginine vasopressin release from supedused hypothalamus and pituitary gland 1 H. Wagner, H. Haberle,
v.
Maier*, and R.E. Lang*
Sektion Nephrologie und 'Department fUr Endokrinologie und Stoffwechsel des Zentrum fUr Innere Medizin und Kinderheilkunde der Universitiit Ulm, Ulm, GFR
ABSTRACT. The study was designed to investigate the effect of various neurotransmitters on the hypothalamus and pituitary gland to determine the sites of their action. Superfused isolated rat hypothalami and pituitary glands demonstrated basal secretion of arginine vasopressin (AVP) and repeated response to stimulation thus showing the viability of the preparation. Acetylcholine and histamine stimulated the release of AVP at the hypothalamic and pituitary levels; dopamine and norepinephrine released AVP in a dose related manner only from the hypothalamus;· angiotensin 11 released AVP in the same fashion only from the pituitary gland. AVP secretion stimulated by dopamine and norepinephrine may represent synaptic inputs which are localized at the hypothalamus and must be distinguish~d from the site of action at the pituitary gland of angiotensin 11. hypothalamus was cut off in a block, using iris scissors. The tissue block was limited rostrally by the anterior border of the optic chiasma, laterally by the hypothalamic fissures, and caudally by the mammillary bodies. The dorsal extent of the cut was to a depth of 2 mm. The explant was gently removed at the cut ophtalmic stalk and was transferred immediately to a plastic vial containing the incubation fluid. Thereafter the entire pituitary gland was gently removed from the sella and was also transferred to a second vial. The medium used for incubation and superfusion resembled cerebrospinal fluid and contained (in mM): NaCI, 126; KCL, 6; NazHP04, 1; MgS04. 7 H20, 0.88; NaHC03, 22; CaCI2, 1.45; O-glucose 0.2%, gassed continously with carbogen (02/C02 = 9515 wtlvol) to pH 7.4, osmolality 298 mosmol. The incubation period lasted 20 min. The tissue, used either as a block or sliced in two halves, was thereafter transferred to superfusion chambers (Millipore no. 10SX0001300). Each plastic chamber, containing three organs in a volume of 0.85 ml, was kept at 37 C in a water bath. Two experiments were run in parallel. A constant flow rate of 0.8 ml/min was maintained by a peristaltic pump (Oesaga, Heidelberg) and, the samples were collected in plastic vials at 2 min intervals. After a 20 min equilibration period, the test substance was added by a sidearm. The stimulation period lasted 10 min. Thereafter a buffer period followed and a further stimulation period was set up. The superfusion period lasted 100 minutes. All collected samples were assayed immediately after the experiment. The AVP content of homogenized hypothalami and pituitary glands was determined after extraction using 0.1 N HCI ethanol. The extract was blown to dryness and assayed; stimulated AVP release was expressed
INTRODUCTION The magnocellular secretory system has been shown to form neurosecretory material in the perikaria of the supraoptic and paraventricular neurons which is transported to the pituitary gland in their axons (1, 29). Immunochemical studies have demonstrated the presence of aminergic and cholinergic fibers in the hypothalamus as well as in the anterior and posterior pituitary gland lobes of the (18, 21). This may lead to the assumption that the secretion of the neurohormones, arginine vasopressin (AVP) and oxytocin, is not only controlled by synaptic inputs at the hypothalamic but also at the hypophysial level. In vitro experiments using the isolated neural lobe (12), the entire hypothalamo-neurohypophysial system (15, 24) and isolated stJpraoptic cells (27) have shown that adrenergic and cholinergic neurons are involved in the AVP release. The purpose of the present study was to investigate the control of AVP release by different putative neurotransmitters at the hypothalamic and 1or the neurohypophysial level. The model of superfused pituitary glands and hypothalami was used to study the dynamics of hormone release.
MATERIALS AND METHODS Male Sprague Oawley rats (120-150 g) were decapitated, the skull was opened and the brain reflected. The
1 Presented
In
part at the XI th Acta Endocrinologica Congress Lausanne, 1977
(37) and part of the thesis of Haberle
Key-words· Arginine vasopressin release, hypophysIs, hypo1halamus. neurotransmitters. superfuslon Correspondence- Or. Hans Wagner, Steinhbvelstr. 9. 0-79 Ulm/Oonau Received September 24. 1977: accepted May 2. 1978
215
H. Wagner, M. Haber/e, V. Maier, and RE Lang
as percent of the basal secretion. Baseline AVP release was determined between the 20th and 40th min. The hypothalami and pituitary glands were examined, in some experiments, by light microscopy for completeness and integrity before and after superfusion.
12.0 ± 1.8 j.lU/organ/1 0 min. The AVP release from a whole compared with two halves of a hypothalamus and pituitary gland was similiar. The AVP extracted from organs, before as well as after the superfusion, amounted to 12.8 mU per hypothalamus and 320 mU per pituitary gland. This shows that the basal release of AVP from the hypothalamic area over 10 minutes is about twenty times higher than that of the pituitary (Table 1) when expressed as % of total hormone content, indicating that the hypothalamus has more hormone in a "releasable form".
Arginine vasopressin radioimmunoassay
200 j.l1 antibody (final dilution 1 : 20,000) was incubated with 100 j.l1 ' 25 1 arginine vasopressin (5000 cpm) and 100 j.l1 standard or superfusion fluid for 24 h; thereafter 50 j.l1 second antibody (anti-rabbit-globuline forte, Behring Frankfurt) was added and after a second 24 h period the incubate was separated by centrifugation (2000 g) for 10 min (36) . Drugs used in the studies were obtained commercially: norepinephrine, dopamine, acetylcholine and nicotine (Fluka AG - Buchs, Switzerland); angiotensin and phentolamine (Ciba Geigy, Wehr). Synthetic arginine vasopressin was a gift from Or. J. Mulder, (Ferring AB Malmo, Sweden).
Hypotha/amus
Stimulations of superfused hypothalami were performed, after a preincubation time of 20 minutes and a further equilibration period of 12 - 20 minutes, with acetylcholine (ACH), norepinephrine (NA), dopamine (DA), histamine (HA), serotonin (OHTA) and angiotensin II (A 11). The time of exposure lasted 10 minutes and during this period the maximum of the AVP release was obtained after 2 - 4 minutes (Fig. 1). During prolonged exposure to the drugs the secretion returned to basal values after 10 - 14 minutes, while stimulation was continued (Fig. 3). After a control period of 60 minutes a repeated stimulation yielded identical peaks over a 120 min. superfusion period (Fig. 2). Significance of the results was tested by the Student's t test and significance was defined as p
