- Email: [email protected]
Synergistic interactions between footshocks and non-osmotic hypovolemia on vasopressin secretion in rats
140
Brain Research, 410 (1987) 140-142 Elsevier
BRE 22227
Synergistic interactions between footshocks and non-osmotic hypovolemia on vasopressin secretion in rats K a t s u e i S h i b u k i 1, T a t s u s h i O n a k a I , M i t s u k o H a m a m u r a 1, Kinji Y a g i I , S a n - e I s h i k a w a 2, Toshikazu Saito 2 and Sho Yoshida 2 1Department of Physiology and 2Departmentof Endocrinology and Metabolism, Jichi MedicalSchool, Tochigi (Japan) (Accepted 20 January 1987) Key words: Footshock; Hypovolemia;Osmotic stimulation; Rat; Synergism;Vasopressin
The effects of i.p. injected hypertonic NaCI and polyethylene glycolon the magnitude of increase in plasma vasopressin after footshocks were studied in male rats, to determine whether hypovolemiaand body fluid osmolality interact with noxious stimuli on vasopressin secretion. Present data have demonstrated that non-osmotic hypovolemiabut not body fluid hyperosmolarity interact significantly and synergisticallywith footshocks to potentiate vasopressinsecretion. Vasopressin (VP)-secreting neurosecretory cells in the rat supraoptic nucleus receive excitatory synaptic inputs after a variety of noxious stimuli 3. Repetitively applied electric footshocks (FS) have been shown to increase plasma VP in rats 6. The previous electrophysiological experiments 9 have demonstrated that excitatory synaptic inputs to supraoptic VP-secreting cells after peripheral nerve stimulation are synergistically augmented under the condition of non-osmotic hypovolemia, but not of increased body fluid osmolality, and thus suggested the possibility that noxious stimuli interact with hypovolemic, but not with hypertonic, stimuli to produce synergistic potentiation of VP secretion by the posterior pituitary. The present study aimed at testing this hypothesis by experiments with radioimmunoassay (RIA) of plasma VP. Seventy-two male rats of the Wistar strain weighing between 190 and 236 g were divided into 9 groups, each of which included 8 rats. Electroconductive cream for electrocardiographic use was painted on the paws. Electric footshocks (10-ms pulses of 0-, 1.5- or 2.0-mA intensity repeated at 50 Hz for 60 s) were applied to each rat through stainless-steel grids of the floor of a box made of lucite
glass (40 × 40 x 60 cm in depth, width and height). To produce an acute non-osmotic hypovolemia according to Dunn et al.l, rats were injected i.p. at a dose of 20 ml/kg b. wt. with 20% polyethylene glycol (PEG, average mol. wt. of 3000, Wako Pure Chemical) dissolved in 0.9% NaCI solution. To increase body fluid osmolality rats were injected i.p. with 9% NaCI at a dose of 5 ml/kg. The interval between the injection and FS was one hour. One minute after termination of the FS rats were decapitated and the trunk blood was collected in a heparinized beaker. Plasma was separated from the sampled blood by •centrifugation for 15 min at 3000 rpm and 4 °C. A 1ml aliquot of plasma was extracted with 2 ml of acetone and 5 ml of diethyl ether and dried in a centrifuge evaporator (RD-31, Yamato), according to Robertson et al. 7. To determine plasma VP by RIA each dried sample was dissolved in 0.5 ml of buffer solution containing 0.1% bovine serum albumin (RIA grade, Fraction V, Sigma), 50 mM barbital sodium adjusted to pH 8.6 with HCI, 10 mM Na2-EDTA and 0.1% sodium azide. Samples (0.2 ml x 2 for each) were mixed with 0.05 ml of rabbit anti-8-arginine vasopressin antiserum (UCB Bioproducts, no. 290235) at 15,000
Correspondence: T. Onaka, Department of Physiology,Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan. 0006-8993/87/$03.50© 1987 Elsevier SciencePublishers B.V. (BiomedicalDivision)
141 times dilution. After 24 h incubation at 4 °C, each sample was mixed with 1000 cpm of [1251]arginine vasopressin (NEX-128, New England Nuclear) dissolved in 0.05 ml of buffer solution. After an additional incubation for 48 h at 4 °C, 0. I ml of 1% bovine y-globulin (Cohn's Fraction II, Sigma) and 1 ml of 25% P E G of an average molecular weight of 7500 (Wako Pure Chemical) were added to the sample, mixed thoroughly and centrifuged. Radioactivity of the precipitate was determined by a gamma counter (ARC-600, Aloka). The assayable range of this R I A system was between 1.0 and 1000 pg of 8-arginine vasopressin (Sigma, 367 IU/mg) per milliliter plasma, and the estimated VP levels were well within this range (1.7-310 pg/ml). VP contents of all samples were determined in a single assay. To determine the effect of P E G or a hypertonic NaCI injection on plasma osmolality and blood hemoglobin concentration (Hb), another 19 rats were anesthetized with diethyl ether and the blood was collected by cardiac puncture. Hb was measured by the cyanmethemoglobin method (Hb-test kit, W a k o Pure Chemical) with a spectrophotometer (100-10, Hitachi). Plasma osmolality was determined by an osmometer (330D, Fiske). An i.p. injection of hypertonic NaCI significantly increased both Hb and plasma osmolality (Table I). On the other hand, i.p. injected P E G significantly increased Hb but not plasma osmolality. These data show that i.p. injected P E G produced an acute nonosmotic hypovolemia in the present experiments. In the rats which received 0-mA FS the treatment with both hypertonic NaCI and P E G significantly increased plasma VP as shown in Fig. 1A. Fig. 1B represents the difference in mean plasma
TABLE I Hemoglobin concentration and plasma osmolality after an i.p. injection of PEG or hypertonic NaCI
Values are means + S.E.M. Treatment
n
Hernoglobin (g/dl)
Osmolality (m Osm/kg H,O)
None PEG NaCI
5 8 6
12.9 _+0.2 14.5 + 0.2* 13.8 + 0.1"
316 + 1 319 + 1 326 + 1"
* Statistically significant (P < 0.005, Mann-Whitney U-test) as compared with the data of uninjected control rats.
A
A
200
r-q NONE r-q ~c,
I
PEG O. v
150 r
10O 0 m m
50
m
E
o~ m Q.
--~-L7
B---
~so [
a.
100 i
~:~
0
,
1.5
f13.8.
E
e-
~5
50
L3 - -1~
Footshock intensity (mA)
Fig. 1. A: plasma VP levels after FS in uninjected control (NONE), hypertonic NaCI-injected (NaCI) and PEG-injected (PEG) rats. Each column represents the range of mean + S.E.M. ** Significance at P < 0.005 (Mann-Whitney U-test) for the data as compared with mean VP in uninjected control rats. B: difference of mean plasma VP between FS-received and uninjected rats. *P < 0.02, **P < 0.005 (Mann-Whitney U-test).
VP between shocked and unshocked rats. Post-hoc comparison of the data suggest that FS with 1.5-mA intensity did not significantly increase plasma VP but FS with 2.0-mA intensity did in uninjected, NaCl-injected and PEG-injected rats ( M a n n - W h i t n e y Utest). Data of whole series of experiments are represented in Fig. 1A. Analyses of variance ( A N O V A ) were applied to all these data sets and the following conclusions were supported: FS significantly increased plasma VP (P < 0.005); i.p. injected P E G increased plasma VP (P < 0.005) and synergistically modulated the potentiating effect of FS on VP secretion (P < 0.05), as compared with uninjected control rats. On the other hand, neither the difference in
142 plasma VP nor the modulatory effects of NaCI on plasma VP after FS were significant (P > 0.05) between uninjected and NaCl-injected rats, although mean VP levels were higher in NaCl-injected than in uninjected control rats. The difference in plasma VP between NaC1- and PEG-injected rats was significant (P < 0.05). These data therefore support the hypothesis that an acute hypovolemia, but not an increase in body fluid osmolality, interacts synergistically with noxious stimuli to potentiate VP secretion by the posterior pituitary. Since the classical work done by Verney l° many studies have shown that an increase in body fluid osmolality potentiates secretion of antidiuretic hormone (VP) by the posterior pituitary. Later studies have demonstrated that hypovolemic or hypotensive stimuli facilitate VP secretion 2'8. In addition to these 'classical' stimuli noxious stimuli have also been shown to increase plasma VP 5,6 and to produce excitatory synaptic inputs in VP-secretory cells 3. Although FS with 0.5 Hz have failed to significantly influence plasma VP level 4, the apparent discrepancy in the effect of FS on plasma VP has been demonstrated to be due to the frequency of FS pulses 6. In the present experiments FS significantly increased plasma VP in an FS intensity-dependent manner
1 Dunn, F.L., Brennan, T.J., Nelson, A.E. and Robertson, G.L., The role of blood osmolalityand volume in regulating vasopressin secretion in the rat, J. Clin. Invest., 52 (1973) 3212-3219. 2 Ginsburg, M., The secretion of antidiuretic hormone in response to haemorrhage and the fate of vasopressin in adrenalectomized rats, J. Endocrinol., 11 (1954) 165-176. 3 Hamamura, M., Shibuki, K. and Yagi, K., Noxious inputs to supraoptic neurosecretory cells in the rat, Neurosci. Res., 2 (1984) 49-61. 4 Knepel, W., Nuno, D. and Hertting, G., Evidence for inhibition by fl-endorphin of vasopressin release during foot shock-induced stress in the rat, Neuroendocrinology, 34 (1982) 353-356. 5 Mirsky, I.A., Stein, M. and Paulisch, G., The secretion of an antidiuretic substance into the circulation of rats exposed to noxious stimuli, Endocrinology, 54 (1954) 491-505.
(ANOVA test, P < 0.005). Concerning the interactions between noxious stimuli and the other two groups of stimuli which potentiate VP secretion, our previous electrophysiological study 9 has suggested that an acute hypovolemia but not an increase in body fluid osmolality synergistically potentiate FS-induced VP secretion. In the present experiments i.p. injected hypertonic NaCI increased not only plasma osmolality but also Hb although the increase in Hb was significantly less than that after PEG injection (Mann-Whitney U-test, P < 0.02). Nevertheless analyses of variance on the present data did not support the hypothesis that FS and NaCI injection synergistically interact on VP secretion. On the other hand the present experiments have clearly demonstrated that FS-induced VP secretion is synergistically potentiated in PEG-injected rats. Thus the present data, together with the previous electrophysiological data 9, lead us to conclude that noxious stimuli interact selectively and synergistically with hypovolemic but not hypertonic stimuli. The authors are indebted to Dr. G. Leng for critical reading of the manuscript in its early stage. This work was supported by the Ministry of Education, Science and Culture, Japan.
60naka, T., Hamamura, M. and Yagi, K., Potentiation of vasopressin secretion by footshocks in rats, Jpn. J. Physiol., 36 (1986) 1253-1260. 7 Robertson, G.L., Mahr, E.A., Athar, S. and Sinha, T., Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Invest., 52 (1973)2340-2352. 8 Share, E., Effects of carotid occlusion and left atrial distention on plasma vasopressin titer, Am. J. Physiol., 208 (1965) 219-223. 9 Shibuki, K. and Yagi, K., Synergistic activation of rat supraoptic neurosecretory neurons by noxious and hypovolemic stimuli, Exp. Brain Res., 62 (1986) 572-578. 10 Verney, E.B., The antidiuretic hormone and the factors which determine its release, Proc. R. Soc. London Set. B, 135 (1947) 25-106.
Brain Research, 410 (1987) 140-142 Elsevier
BRE 22227
Synergistic interactions between footshocks and non-osmotic hypovolemia on vasopressin secretion in rats K a t s u e i S h i b u k i 1, T a t s u s h i O n a k a I , M i t s u k o H a m a m u r a 1, Kinji Y a g i I , S a n - e I s h i k a w a 2, Toshikazu Saito 2 and Sho Yoshida 2 1Department of Physiology and 2Departmentof Endocrinology and Metabolism, Jichi MedicalSchool, Tochigi (Japan) (Accepted 20 January 1987) Key words: Footshock; Hypovolemia;Osmotic stimulation; Rat; Synergism;Vasopressin
The effects of i.p. injected hypertonic NaCI and polyethylene glycolon the magnitude of increase in plasma vasopressin after footshocks were studied in male rats, to determine whether hypovolemiaand body fluid osmolality interact with noxious stimuli on vasopressin secretion. Present data have demonstrated that non-osmotic hypovolemiabut not body fluid hyperosmolarity interact significantly and synergisticallywith footshocks to potentiate vasopressinsecretion. Vasopressin (VP)-secreting neurosecretory cells in the rat supraoptic nucleus receive excitatory synaptic inputs after a variety of noxious stimuli 3. Repetitively applied electric footshocks (FS) have been shown to increase plasma VP in rats 6. The previous electrophysiological experiments 9 have demonstrated that excitatory synaptic inputs to supraoptic VP-secreting cells after peripheral nerve stimulation are synergistically augmented under the condition of non-osmotic hypovolemia, but not of increased body fluid osmolality, and thus suggested the possibility that noxious stimuli interact with hypovolemic, but not with hypertonic, stimuli to produce synergistic potentiation of VP secretion by the posterior pituitary. The present study aimed at testing this hypothesis by experiments with radioimmunoassay (RIA) of plasma VP. Seventy-two male rats of the Wistar strain weighing between 190 and 236 g were divided into 9 groups, each of which included 8 rats. Electroconductive cream for electrocardiographic use was painted on the paws. Electric footshocks (10-ms pulses of 0-, 1.5- or 2.0-mA intensity repeated at 50 Hz for 60 s) were applied to each rat through stainless-steel grids of the floor of a box made of lucite
glass (40 × 40 x 60 cm in depth, width and height). To produce an acute non-osmotic hypovolemia according to Dunn et al.l, rats were injected i.p. at a dose of 20 ml/kg b. wt. with 20% polyethylene glycol (PEG, average mol. wt. of 3000, Wako Pure Chemical) dissolved in 0.9% NaCI solution. To increase body fluid osmolality rats were injected i.p. with 9% NaCI at a dose of 5 ml/kg. The interval between the injection and FS was one hour. One minute after termination of the FS rats were decapitated and the trunk blood was collected in a heparinized beaker. Plasma was separated from the sampled blood by •centrifugation for 15 min at 3000 rpm and 4 °C. A 1ml aliquot of plasma was extracted with 2 ml of acetone and 5 ml of diethyl ether and dried in a centrifuge evaporator (RD-31, Yamato), according to Robertson et al. 7. To determine plasma VP by RIA each dried sample was dissolved in 0.5 ml of buffer solution containing 0.1% bovine serum albumin (RIA grade, Fraction V, Sigma), 50 mM barbital sodium adjusted to pH 8.6 with HCI, 10 mM Na2-EDTA and 0.1% sodium azide. Samples (0.2 ml x 2 for each) were mixed with 0.05 ml of rabbit anti-8-arginine vasopressin antiserum (UCB Bioproducts, no. 290235) at 15,000
Correspondence: T. Onaka, Department of Physiology,Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan. 0006-8993/87/$03.50© 1987 Elsevier SciencePublishers B.V. (BiomedicalDivision)
141 times dilution. After 24 h incubation at 4 °C, each sample was mixed with 1000 cpm of [1251]arginine vasopressin (NEX-128, New England Nuclear) dissolved in 0.05 ml of buffer solution. After an additional incubation for 48 h at 4 °C, 0. I ml of 1% bovine y-globulin (Cohn's Fraction II, Sigma) and 1 ml of 25% P E G of an average molecular weight of 7500 (Wako Pure Chemical) were added to the sample, mixed thoroughly and centrifuged. Radioactivity of the precipitate was determined by a gamma counter (ARC-600, Aloka). The assayable range of this R I A system was between 1.0 and 1000 pg of 8-arginine vasopressin (Sigma, 367 IU/mg) per milliliter plasma, and the estimated VP levels were well within this range (1.7-310 pg/ml). VP contents of all samples were determined in a single assay. To determine the effect of P E G or a hypertonic NaCI injection on plasma osmolality and blood hemoglobin concentration (Hb), another 19 rats were anesthetized with diethyl ether and the blood was collected by cardiac puncture. Hb was measured by the cyanmethemoglobin method (Hb-test kit, W a k o Pure Chemical) with a spectrophotometer (100-10, Hitachi). Plasma osmolality was determined by an osmometer (330D, Fiske). An i.p. injection of hypertonic NaCI significantly increased both Hb and plasma osmolality (Table I). On the other hand, i.p. injected P E G significantly increased Hb but not plasma osmolality. These data show that i.p. injected P E G produced an acute nonosmotic hypovolemia in the present experiments. In the rats which received 0-mA FS the treatment with both hypertonic NaCI and P E G significantly increased plasma VP as shown in Fig. 1A. Fig. 1B represents the difference in mean plasma
TABLE I Hemoglobin concentration and plasma osmolality after an i.p. injection of PEG or hypertonic NaCI
Values are means + S.E.M. Treatment
n
Hernoglobin (g/dl)
Osmolality (m Osm/kg H,O)
None PEG NaCI
5 8 6
12.9 _+0.2 14.5 + 0.2* 13.8 + 0.1"
316 + 1 319 + 1 326 + 1"
* Statistically significant (P < 0.005, Mann-Whitney U-test) as compared with the data of uninjected control rats.
A
A
200
r-q NONE r-q ~c,
I
PEG O. v
150 r
10O 0 m m
50
m
E
o~ m Q.
--~-L7
B---
~so [
a.
100 i
~:~
0
,
1.5
f13.8.
E
e-
~5
50
L3 - -1~
Footshock intensity (mA)
Fig. 1. A: plasma VP levels after FS in uninjected control (NONE), hypertonic NaCI-injected (NaCI) and PEG-injected (PEG) rats. Each column represents the range of mean + S.E.M. ** Significance at P < 0.005 (Mann-Whitney U-test) for the data as compared with mean VP in uninjected control rats. B: difference of mean plasma VP between FS-received and uninjected rats. *P < 0.02, **P < 0.005 (Mann-Whitney U-test).
VP between shocked and unshocked rats. Post-hoc comparison of the data suggest that FS with 1.5-mA intensity did not significantly increase plasma VP but FS with 2.0-mA intensity did in uninjected, NaCl-injected and PEG-injected rats ( M a n n - W h i t n e y Utest). Data of whole series of experiments are represented in Fig. 1A. Analyses of variance ( A N O V A ) were applied to all these data sets and the following conclusions were supported: FS significantly increased plasma VP (P < 0.005); i.p. injected P E G increased plasma VP (P < 0.005) and synergistically modulated the potentiating effect of FS on VP secretion (P < 0.05), as compared with uninjected control rats. On the other hand, neither the difference in
142 plasma VP nor the modulatory effects of NaCI on plasma VP after FS were significant (P > 0.05) between uninjected and NaCl-injected rats, although mean VP levels were higher in NaCl-injected than in uninjected control rats. The difference in plasma VP between NaC1- and PEG-injected rats was significant (P < 0.05). These data therefore support the hypothesis that an acute hypovolemia, but not an increase in body fluid osmolality, interacts synergistically with noxious stimuli to potentiate VP secretion by the posterior pituitary. Since the classical work done by Verney l° many studies have shown that an increase in body fluid osmolality potentiates secretion of antidiuretic hormone (VP) by the posterior pituitary. Later studies have demonstrated that hypovolemic or hypotensive stimuli facilitate VP secretion 2'8. In addition to these 'classical' stimuli noxious stimuli have also been shown to increase plasma VP 5,6 and to produce excitatory synaptic inputs in VP-secretory cells 3. Although FS with 0.5 Hz have failed to significantly influence plasma VP level 4, the apparent discrepancy in the effect of FS on plasma VP has been demonstrated to be due to the frequency of FS pulses 6. In the present experiments FS significantly increased plasma VP in an FS intensity-dependent manner
1 Dunn, F.L., Brennan, T.J., Nelson, A.E. and Robertson, G.L., The role of blood osmolalityand volume in regulating vasopressin secretion in the rat, J. Clin. Invest., 52 (1973) 3212-3219. 2 Ginsburg, M., The secretion of antidiuretic hormone in response to haemorrhage and the fate of vasopressin in adrenalectomized rats, J. Endocrinol., 11 (1954) 165-176. 3 Hamamura, M., Shibuki, K. and Yagi, K., Noxious inputs to supraoptic neurosecretory cells in the rat, Neurosci. Res., 2 (1984) 49-61. 4 Knepel, W., Nuno, D. and Hertting, G., Evidence for inhibition by fl-endorphin of vasopressin release during foot shock-induced stress in the rat, Neuroendocrinology, 34 (1982) 353-356. 5 Mirsky, I.A., Stein, M. and Paulisch, G., The secretion of an antidiuretic substance into the circulation of rats exposed to noxious stimuli, Endocrinology, 54 (1954) 491-505.
(ANOVA test, P < 0.005). Concerning the interactions between noxious stimuli and the other two groups of stimuli which potentiate VP secretion, our previous electrophysiological study 9 has suggested that an acute hypovolemia but not an increase in body fluid osmolality synergistically potentiate FS-induced VP secretion. In the present experiments i.p. injected hypertonic NaCI increased not only plasma osmolality but also Hb although the increase in Hb was significantly less than that after PEG injection (Mann-Whitney U-test, P < 0.02). Nevertheless analyses of variance on the present data did not support the hypothesis that FS and NaCI injection synergistically interact on VP secretion. On the other hand the present experiments have clearly demonstrated that FS-induced VP secretion is synergistically potentiated in PEG-injected rats. Thus the present data, together with the previous electrophysiological data 9, lead us to conclude that noxious stimuli interact selectively and synergistically with hypovolemic but not hypertonic stimuli. The authors are indebted to Dr. G. Leng for critical reading of the manuscript in its early stage. This work was supported by the Ministry of Education, Science and Culture, Japan.
60naka, T., Hamamura, M. and Yagi, K., Potentiation of vasopressin secretion by footshocks in rats, Jpn. J. Physiol., 36 (1986) 1253-1260. 7 Robertson, G.L., Mahr, E.A., Athar, S. and Sinha, T., Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Invest., 52 (1973)2340-2352. 8 Share, E., Effects of carotid occlusion and left atrial distention on plasma vasopressin titer, Am. J. Physiol., 208 (1965) 219-223. 9 Shibuki, K. and Yagi, K., Synergistic activation of rat supraoptic neurosecretory neurons by noxious and hypovolemic stimuli, Exp. Brain Res., 62 (1986) 572-578. 10 Verney, E.B., The antidiuretic hormone and the factors which determine its release, Proc. R. Soc. London Set. B, 135 (1947) 25-106.