- Email: [email protected]
Chapter 56: Progressive increases of protein synthesis in the circumventricular organs during chronic dehydration in rats
A. Ermisch, R . Landgraf and H.-J. Riihle (Eds.) Progress in Brain Reswrch, Vol. 91
0 1992 Elsevier Science Publishers
B.V. All
435
rights reserved
CHAPTER 56
Progressive increases of protein synthesis in the circumventricular organs during chronic dehydration in rats Patrick Lepetit, Eric Grange, Nadine Gay and Pierre Bobillier INSERM U171, CNRS UA1195, Groupe de Neurochimie Fonctionnelle, HGpital Lyon Sud, 69310 Pierre Bdnite, France, and CNRS URAll95, Laboratoire d’Anatomie Pathologique, Facultd de Mddicine Alexis Carrel, 69372 L YON Cedex 08, France
The quantitative autoradiographic method with L(35S)methioninewas applied to investigate the effect of chronic dehydration on rates of protein synthesis in circumventricular organs (CVOs). Water deprivation for 1,2 and 3 days causes progressive increases of protein synthesis in the subfornical organ (SFO),the area postrema, the organum vasculosum laminae terminalis and the neurohypophysis. Chronic salt ingestion with 2% NaCl in drinking water for 3 days resulted in increases of protein synthesis in the CVOs similar to those found after 3 days water deprivation, with only one exception, the SFO, in which the rise
in protein synthesis was of lower amplitude after 3 days salt ingestion as compared to 3 days water deprivation. These results suggest that several circulating factors related to intracellular dehydration and the high plasma levels of the neurohormones vasopressin and oxytocin are probably important determinants of the rise of protein synthesisincircumventricular organs. Alternatively, the elevated level of blood-borne angiotensin I1 may well explain the higher metabolic response of the SFO following water deprivation compared to salt ingestion.
Introduction
utilization (Gross et al., 1985), RNA and protein synthesis (George, 1974; Summy-Long and Severs, 1979; Lepetit et al., 1988). During water deprivation (WD), plasma osmolality, vasopressin and oxytocin secretion, and the formation of blood-borne angiotensin I1 increase; these peripheral stimuli may directly affect cellular activity in the CVOs. Alternatively, chronic salt ingestion (SI) with 2% NaCl in drinking water is a model of intracellular dehydration, which does not involve the peripheral renineangiotensin system but induces similar increases in plasma osmolality and vasopressin secretion (Jones and Pickering, 1969). Therefore, to better differentiate what type of circulating factors may initiate a metabolic response in vivo, it was of interest to compare the effects of 1,2 and 3 days WD and 3 days SI, on rates of protein synthesis in the CVOs. Local rates of protein synthesis were estimated by quan-
It is now well established that the subfornical organ (SFO), the organum vasculosum laminae terminalis (OVLT) and the area postrema (AP) are parts of a complex network of cerebral structures involved in the regulation of salt-water balance and neuroendocrine function. In addition, these circumventricular organs (CVOs), deficient in the blood-brain barrier, contain binding sites for several circulating hormones (i.e., angiotensin I1 and atrial natriuretic factor) which do not normally enter the brain from plasma. These particular features of CVOs suggest that cells within CVOs may be specifically implicated in the central detection of circulating hormones (Ermisch et al., 1985). Previous reports have indicated that chronic dehydration increases the metabolic’ activity of the SFO, with elevated glucose
436
titative autoradiographic measurement of the incorporation of plasma methionine into proteins, in freely moving rats as previously described (Lestage et al., 1987; Lepetit et al., 1988).
Methods Male Sprague-Dawley rats (200 - 240 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.), 7 days prior to the experiment for the implantation of arterial and venous cannulae. On the fourth day after surgery, at about 2 p.m., rats were weighed and the water bottles were replaced by: (1) tap water (control); (2) a 2% (w/v) NaCl solution (SI), for 3 days; and (3) no bottle (water deprived), for 1,2 and
3 days. All rats were housed in the same conditions with free access to food, under conditions of controlled lighting (12 h/12 h) and temperature (24°C). The method for the measurement of local rates of methionine incorporation into brain proteins have been previously described in detail (Lestage et al., 1987). Briefly, it is based on the use of ~ - ( ~ ~ S ) m e thionine as a tracer to measure the exchange of methionine between plasma and tissue and its incorporation into proteins. By mathematical analysis of the kinetics of exchanges of methionine between these three compartments an operational equation was derived that defines the rate of methionine incorporation in terms of the time course of the specific activity of methionine in plasma, the final brain
Fig. I . Autoradiographs of coronal brain sections (20 am thick) 1 h after the intravenous injection of ~-(%)metbionine from control (a, d, top) and water-deprived rats (b, c, d, bottom) at the level of the choroid plexus (CP), SFO, OVLT and pituitary: neurohypophysis (NH) and adenohypophysis (AH). The figures show high concentration of the label in the choroid plexus, SFO, OVLT, adenohypophysis and supraoptic nucleus. (Bars, 1 mm.)
437
35S concentration, and experimentally determined rate constants. The rate constants used for cerebral grey matter were applied to estimate the rates of methionine incorporation into proteins in the CVOs . c 5 For the determination of the time course of plas.5 4' ma methionine specific activity, 16 sequential arte..rial blood samples (50 pl, followed by physiological 5 3' saline to replace blood loss) were taken during 60 ; 2' min after the i.v. pulse of ~ - ( ~ ~ S ) m e t h i o n i n e.-C .o 1 ' (specific activity > 1000Ci/mmol; 400 pCi/kg) was 5 0' delivered at about 2 p.m. The plasma samples were Pn AH NH OVLT SFO AP deproteinized and 35S radioactivity was measured Fig. 2. Effect of 1, 2 and 3 days water deprivation (WD) and 3 by liquid scintillation counting. One hour after the days salt ingestion (SI) on methionine utilization in circumvenpulse, the rats were killed by an overdose of pentricular organs and pituitary: pineal (Pn), adenohypophysis (AH), neurohypophysis (NH), organum vasculosum laminae tertobarbital (250 mg/kg) and brains were prepared for minalis (OVLT), subfornical organ (SFO) and area postrema the quantitative autoradiographic measurement of (AP). Values are averages f S.E.M. and are expressed as nmol/g local 35S concentration in individual CVOs. From per minute (n = 5 - 7). the time courses of methionine-specific activity in * P < 0.05 as compared to controls (n = 6); " P < 0.01 as complasma and local 35S concentration in CVOs local pared to 3 days WD (n = 5) with Bonferroni f-test. rates of methionine incorporation into protein were blood at the end of the kinetic study together with calculated by means of the operational equation of the blood volume of individual CVOs reported by the method (Lestage et al., 1987). Gross et al. (1987). Finally, blood contamination All values are given as averages k S.E.M. Data represents less than 3% (pineal), 5('10 (SFO, adenowere statistically analyzed, for multiple comparihypophysis) and 8% (OVLT, neurohypophysis) of sons, by the Bonferroni t-test. the total 35Sradioactivity in CVOs. Because the rats Results were sacrificed 1 h after the injection of tracer, any contribution to the 35S concentration in the neurohypophysis of labeled neurosecretory material Representative autoradiographs obtained from control and water deprived rats are illustrated in Fig. transported intracellularly may be eliminated in view of the fast transport rate previously reported in 1, showing high concentrations of 35S in the SFO, OVLT, adenohypophysis, choroi'd plexus and supthe hypothalamo-neurohypophyseal system (1 mm/h). Among CVOs and pituitary, the highest raoptic nucleus of the hypothalamus. The results of rates of methionine utilization are found in the the quantitative analysis of autoradiographs and pineal and adenohypophysis, followed by the SFO kinetics are summarized in Fig. 2; they indicate that WD progressively increases protein synthesis in and AP. The levels of methionine utilization are SFO, OVLT, AP and neurohypophysis. The commuch lower in the OVLT and neurohypophysis. Methionine utilization in the SFO, OVLT and A P parison between 3 days WD and 3 days SI shows that the two treatments have similar effects in the OVLT, primarily reflects biosynthetic activity of neuronal, glial and endothelial cells; whereas, in the AP, neurohypophysis and pineal, whereas, the increase of protein synthesis in the SFO is of lower neurohypophysis, this index may well reflect the amplitude after 3 days SI compared to 3 days WD. biosynthetic activity of pituicytes or endothelial cells. However, the cellular uptake of labeled proThe importance of blood contamination in CVOs was estimated from the total 35S radioactivity in teins from plasma (i.e., albumin, angiotensinogen)
-
438
may obscure the data presented here. This contribution is likely to be small, but at present we cannot differentiate whether changes in the uptake and retention of circulating labeled proteins bring significant contribution to the apparent rise of protein synthesis following WD and SI.
Ute to the increased protein synthesis in CVOs following chronic dehydration. Finally, these results provide evidence with suggests that the progressive rise of protein synthesis in CVOs during dehydration reveals a progressive increase of their functional capacity related to changes in plasma composition (osmolality, sodium and hormones).
Discussion The present study provides, for the first time, quantitative estimates of the effect of water deprivation and salt ingestion on rates of protein synthesis in circumventricular organs. The rise of protein synthesis in the SFO is of major importance after 3 days of WD. This change is consistent with the previously reported increase of RNA (George, 1974; SummyLong and Severs, 1979) and protein synthesis (Santer et al., 1986; Lepetit et al., 1988) in the SFO following chronic dehydration. The particular importance of the SFO in mediating the effects of bloodborne angiotensin I1 on thirst and antidiuresis suggests that the progressive elevation of circulating levels of angiotensin I1 during W D may initiate a proportional metabolic response in the SFO (Gross et al., 1985). Since the significant elevation of protein synthesis in the SFO following 3 days of SI is unlikely to be initiated by circulating angiotensin 11, it is expected that elevated levels of angiotensin 11 may only explain the higher metabolic response of the SFO following 3 days WD compared to 3 days SI. It is of interest to consider that 3 days WD and 3 days SI which similarly increase plasma osmolality, vasopressin and oxytocin secretion (Jones and Pickering, 1969) induce similar changes of protein synthesis in the AP, OVLT and neurohypophysis. This indicates that several other factors such as hypertonicity of plasma, circulating vasopressin, oxytocin and atrial natriuretic factor are also liable to initiate changes of the protein biosynthetic activities in these CVOs. Considering the major elevation of plasma vasopressin and oxytocin levels following 3 days WD and 3 days SI, it could be hypothesized that these two neurohormones may contrib-
Acknowledgements This work was supported by grants from INSERM (U171, CRE: 896002) and CNRS (URA1195).
References Ermisch, A., Riihle, H.J., Landgraf, R. and Hess, J. (1985) Blood-brain barrier and peptides. J . Cereb. Blood Flow Merab., 5 : 350-357. George, J.M. (1974) Hypothalamic sites of incorporation of [3H]cytidine into RNA in response to oral hypertonic saline. Brain Res., 73: 184- 187. Gross, P.M., Kadekaro, M., Sokoloff, L.. Holcomb, H.H. and Saavedra, J.M. (1985) Alteration of local cerebral glucose utilization during chronic dehydration in rats. Brain Rex, 330: 329 - 336. Gross, P.M., Blasberg, R.G., Fenstermacher, J.D. and Patlak, C.S. (1987) The microcirculation of rat circumventricular organs and pituitary gland. Brain Res. Bull., 18: 73 - 85. Jones, C.W. and Pickering, B.T. (1969) Comparison of the effects of water deprivation and sodium chloride inhibition on the hormone content of the neurohypophysis of the rat. J . Physiol. (Lond.), 203: 449 - 458. Lepetit, P., Lestage, P., Jouvet, M. and Bobillier, P. (1988) Localization of cerebral protein synthesis alterations in response to water deprivation in rats. Neuroendocrinology, 48: 271 - 279. Lestage, P., Gonon, M., Lepetit, P., Vitte, P.A., Debilly, G . , Rossatto, C., Lecestre, D. and Bobillier, P. (1987) An in vivo kinetic model with ~-~'S-methionine for the determination of local cerebral rates of methionine incorporation into protein in the rat. J. Neurochem., 48: 352-363. Santer, D.M., Heydorn, W.E., Creed, G . J . , Klein, D.C. and Jacobowitz (1986) Subfornical organ: effects of salt loading and water deprivation on in vitro radioamino acid incorporation into individual proteins. Brain Res., 372: 107 - 114. Summy-Long, J.Y. and Severs, W.B. (1979) Macromolecular changes in the subfornical organ area after dehydration and renin. Am. J. Physiol., 237: R26 - R38.
0 1992 Elsevier Science Publishers
B.V. All
435
rights reserved
CHAPTER 56
Progressive increases of protein synthesis in the circumventricular organs during chronic dehydration in rats Patrick Lepetit, Eric Grange, Nadine Gay and Pierre Bobillier INSERM U171, CNRS UA1195, Groupe de Neurochimie Fonctionnelle, HGpital Lyon Sud, 69310 Pierre Bdnite, France, and CNRS URAll95, Laboratoire d’Anatomie Pathologique, Facultd de Mddicine Alexis Carrel, 69372 L YON Cedex 08, France
The quantitative autoradiographic method with L(35S)methioninewas applied to investigate the effect of chronic dehydration on rates of protein synthesis in circumventricular organs (CVOs). Water deprivation for 1,2 and 3 days causes progressive increases of protein synthesis in the subfornical organ (SFO),the area postrema, the organum vasculosum laminae terminalis and the neurohypophysis. Chronic salt ingestion with 2% NaCl in drinking water for 3 days resulted in increases of protein synthesis in the CVOs similar to those found after 3 days water deprivation, with only one exception, the SFO, in which the rise
in protein synthesis was of lower amplitude after 3 days salt ingestion as compared to 3 days water deprivation. These results suggest that several circulating factors related to intracellular dehydration and the high plasma levels of the neurohormones vasopressin and oxytocin are probably important determinants of the rise of protein synthesisincircumventricular organs. Alternatively, the elevated level of blood-borne angiotensin I1 may well explain the higher metabolic response of the SFO following water deprivation compared to salt ingestion.
Introduction
utilization (Gross et al., 1985), RNA and protein synthesis (George, 1974; Summy-Long and Severs, 1979; Lepetit et al., 1988). During water deprivation (WD), plasma osmolality, vasopressin and oxytocin secretion, and the formation of blood-borne angiotensin I1 increase; these peripheral stimuli may directly affect cellular activity in the CVOs. Alternatively, chronic salt ingestion (SI) with 2% NaCl in drinking water is a model of intracellular dehydration, which does not involve the peripheral renineangiotensin system but induces similar increases in plasma osmolality and vasopressin secretion (Jones and Pickering, 1969). Therefore, to better differentiate what type of circulating factors may initiate a metabolic response in vivo, it was of interest to compare the effects of 1,2 and 3 days WD and 3 days SI, on rates of protein synthesis in the CVOs. Local rates of protein synthesis were estimated by quan-
It is now well established that the subfornical organ (SFO), the organum vasculosum laminae terminalis (OVLT) and the area postrema (AP) are parts of a complex network of cerebral structures involved in the regulation of salt-water balance and neuroendocrine function. In addition, these circumventricular organs (CVOs), deficient in the blood-brain barrier, contain binding sites for several circulating hormones (i.e., angiotensin I1 and atrial natriuretic factor) which do not normally enter the brain from plasma. These particular features of CVOs suggest that cells within CVOs may be specifically implicated in the central detection of circulating hormones (Ermisch et al., 1985). Previous reports have indicated that chronic dehydration increases the metabolic’ activity of the SFO, with elevated glucose
436
titative autoradiographic measurement of the incorporation of plasma methionine into proteins, in freely moving rats as previously described (Lestage et al., 1987; Lepetit et al., 1988).
Methods Male Sprague-Dawley rats (200 - 240 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.), 7 days prior to the experiment for the implantation of arterial and venous cannulae. On the fourth day after surgery, at about 2 p.m., rats were weighed and the water bottles were replaced by: (1) tap water (control); (2) a 2% (w/v) NaCl solution (SI), for 3 days; and (3) no bottle (water deprived), for 1,2 and
3 days. All rats were housed in the same conditions with free access to food, under conditions of controlled lighting (12 h/12 h) and temperature (24°C). The method for the measurement of local rates of methionine incorporation into brain proteins have been previously described in detail (Lestage et al., 1987). Briefly, it is based on the use of ~ - ( ~ ~ S ) m e thionine as a tracer to measure the exchange of methionine between plasma and tissue and its incorporation into proteins. By mathematical analysis of the kinetics of exchanges of methionine between these three compartments an operational equation was derived that defines the rate of methionine incorporation in terms of the time course of the specific activity of methionine in plasma, the final brain
Fig. I . Autoradiographs of coronal brain sections (20 am thick) 1 h after the intravenous injection of ~-(%)metbionine from control (a, d, top) and water-deprived rats (b, c, d, bottom) at the level of the choroid plexus (CP), SFO, OVLT and pituitary: neurohypophysis (NH) and adenohypophysis (AH). The figures show high concentration of the label in the choroid plexus, SFO, OVLT, adenohypophysis and supraoptic nucleus. (Bars, 1 mm.)
437
35S concentration, and experimentally determined rate constants. The rate constants used for cerebral grey matter were applied to estimate the rates of methionine incorporation into proteins in the CVOs . c 5 For the determination of the time course of plas.5 4' ma methionine specific activity, 16 sequential arte..rial blood samples (50 pl, followed by physiological 5 3' saline to replace blood loss) were taken during 60 ; 2' min after the i.v. pulse of ~ - ( ~ ~ S ) m e t h i o n i n e.-C .o 1 ' (specific activity > 1000Ci/mmol; 400 pCi/kg) was 5 0' delivered at about 2 p.m. The plasma samples were Pn AH NH OVLT SFO AP deproteinized and 35S radioactivity was measured Fig. 2. Effect of 1, 2 and 3 days water deprivation (WD) and 3 by liquid scintillation counting. One hour after the days salt ingestion (SI) on methionine utilization in circumvenpulse, the rats were killed by an overdose of pentricular organs and pituitary: pineal (Pn), adenohypophysis (AH), neurohypophysis (NH), organum vasculosum laminae tertobarbital (250 mg/kg) and brains were prepared for minalis (OVLT), subfornical organ (SFO) and area postrema the quantitative autoradiographic measurement of (AP). Values are averages f S.E.M. and are expressed as nmol/g local 35S concentration in individual CVOs. From per minute (n = 5 - 7). the time courses of methionine-specific activity in * P < 0.05 as compared to controls (n = 6); " P < 0.01 as complasma and local 35S concentration in CVOs local pared to 3 days WD (n = 5) with Bonferroni f-test. rates of methionine incorporation into protein were blood at the end of the kinetic study together with calculated by means of the operational equation of the blood volume of individual CVOs reported by the method (Lestage et al., 1987). Gross et al. (1987). Finally, blood contamination All values are given as averages k S.E.M. Data represents less than 3% (pineal), 5('10 (SFO, adenowere statistically analyzed, for multiple comparihypophysis) and 8% (OVLT, neurohypophysis) of sons, by the Bonferroni t-test. the total 35Sradioactivity in CVOs. Because the rats Results were sacrificed 1 h after the injection of tracer, any contribution to the 35S concentration in the neurohypophysis of labeled neurosecretory material Representative autoradiographs obtained from control and water deprived rats are illustrated in Fig. transported intracellularly may be eliminated in view of the fast transport rate previously reported in 1, showing high concentrations of 35S in the SFO, OVLT, adenohypophysis, choroi'd plexus and supthe hypothalamo-neurohypophyseal system (1 mm/h). Among CVOs and pituitary, the highest raoptic nucleus of the hypothalamus. The results of rates of methionine utilization are found in the the quantitative analysis of autoradiographs and pineal and adenohypophysis, followed by the SFO kinetics are summarized in Fig. 2; they indicate that WD progressively increases protein synthesis in and AP. The levels of methionine utilization are SFO, OVLT, AP and neurohypophysis. The commuch lower in the OVLT and neurohypophysis. Methionine utilization in the SFO, OVLT and A P parison between 3 days WD and 3 days SI shows that the two treatments have similar effects in the OVLT, primarily reflects biosynthetic activity of neuronal, glial and endothelial cells; whereas, in the AP, neurohypophysis and pineal, whereas, the increase of protein synthesis in the SFO is of lower neurohypophysis, this index may well reflect the amplitude after 3 days SI compared to 3 days WD. biosynthetic activity of pituicytes or endothelial cells. However, the cellular uptake of labeled proThe importance of blood contamination in CVOs was estimated from the total 35S radioactivity in teins from plasma (i.e., albumin, angiotensinogen)
-
438
may obscure the data presented here. This contribution is likely to be small, but at present we cannot differentiate whether changes in the uptake and retention of circulating labeled proteins bring significant contribution to the apparent rise of protein synthesis following WD and SI.
Ute to the increased protein synthesis in CVOs following chronic dehydration. Finally, these results provide evidence with suggests that the progressive rise of protein synthesis in CVOs during dehydration reveals a progressive increase of their functional capacity related to changes in plasma composition (osmolality, sodium and hormones).
Discussion The present study provides, for the first time, quantitative estimates of the effect of water deprivation and salt ingestion on rates of protein synthesis in circumventricular organs. The rise of protein synthesis in the SFO is of major importance after 3 days of WD. This change is consistent with the previously reported increase of RNA (George, 1974; SummyLong and Severs, 1979) and protein synthesis (Santer et al., 1986; Lepetit et al., 1988) in the SFO following chronic dehydration. The particular importance of the SFO in mediating the effects of bloodborne angiotensin I1 on thirst and antidiuresis suggests that the progressive elevation of circulating levels of angiotensin I1 during W D may initiate a proportional metabolic response in the SFO (Gross et al., 1985). Since the significant elevation of protein synthesis in the SFO following 3 days of SI is unlikely to be initiated by circulating angiotensin 11, it is expected that elevated levels of angiotensin 11 may only explain the higher metabolic response of the SFO following 3 days WD compared to 3 days SI. It is of interest to consider that 3 days WD and 3 days SI which similarly increase plasma osmolality, vasopressin and oxytocin secretion (Jones and Pickering, 1969) induce similar changes of protein synthesis in the AP, OVLT and neurohypophysis. This indicates that several other factors such as hypertonicity of plasma, circulating vasopressin, oxytocin and atrial natriuretic factor are also liable to initiate changes of the protein biosynthetic activities in these CVOs. Considering the major elevation of plasma vasopressin and oxytocin levels following 3 days WD and 3 days SI, it could be hypothesized that these two neurohormones may contrib-
Acknowledgements This work was supported by grants from INSERM (U171, CRE: 896002) and CNRS (URA1195).
References Ermisch, A., Riihle, H.J., Landgraf, R. and Hess, J. (1985) Blood-brain barrier and peptides. J . Cereb. Blood Flow Merab., 5 : 350-357. George, J.M. (1974) Hypothalamic sites of incorporation of [3H]cytidine into RNA in response to oral hypertonic saline. Brain Res., 73: 184- 187. Gross, P.M., Kadekaro, M., Sokoloff, L.. Holcomb, H.H. and Saavedra, J.M. (1985) Alteration of local cerebral glucose utilization during chronic dehydration in rats. Brain Rex, 330: 329 - 336. Gross, P.M., Blasberg, R.G., Fenstermacher, J.D. and Patlak, C.S. (1987) The microcirculation of rat circumventricular organs and pituitary gland. Brain Res. Bull., 18: 73 - 85. Jones, C.W. and Pickering, B.T. (1969) Comparison of the effects of water deprivation and sodium chloride inhibition on the hormone content of the neurohypophysis of the rat. J . Physiol. (Lond.), 203: 449 - 458. Lepetit, P., Lestage, P., Jouvet, M. and Bobillier, P. (1988) Localization of cerebral protein synthesis alterations in response to water deprivation in rats. Neuroendocrinology, 48: 271 - 279. Lestage, P., Gonon, M., Lepetit, P., Vitte, P.A., Debilly, G . , Rossatto, C., Lecestre, D. and Bobillier, P. (1987) An in vivo kinetic model with ~-~'S-methionine for the determination of local cerebral rates of methionine incorporation into protein in the rat. J. Neurochem., 48: 352-363. Santer, D.M., Heydorn, W.E., Creed, G . J . , Klein, D.C. and Jacobowitz (1986) Subfornical organ: effects of salt loading and water deprivation on in vitro radioamino acid incorporation into individual proteins. Brain Res., 372: 107 - 114. Summy-Long, J.Y. and Severs, W.B. (1979) Macromolecular changes in the subfornical organ area after dehydration and renin. Am. J. Physiol., 237: R26 - R38.