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Neonatal exposure to vasopressin decreases vasopressin binding sites in the adult kidney
Peptides, Vol. 5, pp. 1217-1219, 1984. ~ Ankho International inc. Printed in the U.S.A.
0196-9781/84 $3.00 + .IX)
BRIEF C O M M U N I C A T I O N
Neonatal Exposure to Vasopressin Decreases Vasopressin Binding Sites in the Adult Kidney G A I L E. H A N D E L M A N N
z AND SAMUEL
C. S A Y S O N
Laboratory o f Developmental Neurobiology, N I C H D - N I H , Bethesda, M D 20205 R e c e i v e d 16 A u g u s t 1984 HANDELMANN, G. E. AND S. C. SAYSON. Neonatal exposure to vasopressin decreases vasopressin binding sites in the adult kidney. PEPTIDES 5(6) 1217-1219, 1984.mPrevious experiments showed that rats injected with vasopressin (AVP) during the first seven days after birth were less sensitive as adults to the antidiuretic effects of AVP than were control rats. In the present experiment, binding sites for AVP were measured in the kidneys of similarly treated adult rats. Neonatal exposure to AVP significantly decreased the number of binding sites in the adults, but did not affect the binding affinity of the sites. It is concluded that neonatal exposure to AVP which produces a long-lasting decrease in responsiveness of the kidney to AVP is correlated with a reduction in the number of AVP binding sites in the tissue. Vasopressin
Vasopressin binding sites
Kidney
Development
IN previous experiments, we showed that exogenously administered vasopressin (AVP), a neurohypophyseal peptide hormone, could influence the development o f one of its target organs, the kidney [5]. Administration of A V P to neonatal rats produced a long-term deleterious effect on the AVP-mediated osmoregulatory function of the kidney. The rats exhibited polyuria as adults, and their kidneys were relatively insensitive to A V P in a bioassay for antidiuresis. This insensitivity was correlated with a deficit in the kidney AVP-receptor mediated formation of cAMP, although parathyroid hormone- and caleitonin-stimulated c A M P formation in this tissue were normal. This deficit could therefore have been due to either a reduced number of A V P receptors, decreased binding efficiency of the receptors, or an impairment of the receptor-adenylate cyclase coupling. In the present experiment, we investigated AVP-reeeptor binding in the kidneys of adult rats treated as neonates with AVP, and we demonstrate that the number of binding sites is reduced in these rats.
buffered saline (PBS), pH 7.4, or A V P (4 ~g/100 g body weight, dissolved in PBS; Calbiochem, 395 U/rag). The assignment of pups within a litter to a treatment group was done at random. The pups were weaned on day 23.
Binding Assay When the rats were at least 90 days old, they were decapitated and their kidneys rapidly removed and frozen on powdered dry ice. The frozen kidneys were cut into 25 micron sections and the sections thaw-mounted on gelatin-coated slides. Sections taken through the largest aspect of the kidney, in which cortex, medulla, and collecting duets were clearly visible, were used for the assay. Adjacent sections were used for protein determination using fluorescamine [1] with bovine serum albumin as the standard. The slides were pre-incubated for 15 minutes at 25°C in 100 mM Tris pH 7.4, containing 0.2% bovine serum albumin, 10 mM MgCI~, 40 ~g/ml bacitracin, 6/~g/ml soybean trypsin inhibitor, and 0.1% lysine. The slides were removed from the preincubation buffer, and 200 p,l o f buffer containing varying amounts of aH-AVP (51 Ci/mmol; New England Nuclear) was placed directly onto the kidney section. The sections were incubated for 30 minutes at 25°C. At the end of the incubation, the slides were placed in racks and transferred sequentially through four one-minute rinses of 50 mM Tris, pH 7.4 at 4°C. Each kidney section was then wiped from the
METHOD
Subjects and Peptide Treatment Two litters of albino rat pups were cross-fostered and the litters culled to 10 pups each. Beginning the day after birth (neonatal day 11 and ending neonatal day 7. each pup received daily subcutaneous injections of either phosphate-
'Requests for reprints should be addressed to Dr. G. E. Handelmann, Lab. of Developmental Neurobiology, NICHD-NIH, Bldg. 36 Rm. 2A21. Bethesda, MD 20205. G.E.H. is a Pharmacology Research Associate of the National Institute of General Medical Sciences.
1217
1218
~
H A N D E L M A N N A N D SAYSON
70
~
I1 1
I 5
I 10
100
I I 20 30 PH] AVP, nmolm/lil
I 40
I 50
FIG. 1. Plot of specific binding of [:~H]AVPto kidney slices taken from adult rats treated as neonates with either AVP or PBS. *Significantly different from Neonatal Saline group (Student's t-test, p<0.05). Each point is the mean of duplicates from six rats,-+S.E.M.
• Neonatal AVP
~ 40
•
o
o
•
o
20 • e I
20
slide with a glass filter (Whatman OF/B), placed in 8 ml of Aquasol (New England Nuclear), vortexed, and the radioactivity counted. Total binding was determined by incubating sections with concentrations of aH-AVP ranging from 0.5 to 50 nM. Nonspecific binding was defined as the amount of radioactivity which remained bound when sections were incubated with 3H-AVP in the presence of 10-6 M unlabelled AVP. RESULTS
Binding Assay The Bma.~ and K,) values were obtained by regression analysis of Scatchard plots of saturation binding data (Fig. I), using a curve-fitting computer program (BRIGHT; [3]). The maximal binding capacity (Bma.O of kidney sections from rats treated as neonates with AVP was about 26% less than that of the control rats (Fig. 2), or 68__.6 and 92+_9 fmols/mg protein, respectively (p<0.05, Student's t-test). The Scatchard plots (Fig. 2) were linear, and indicated that the dissociation constants (K,) for the two groups' binding were similar, The K , for the group treated with AVP was 0.86+_0.06 nmol, and that for the control group was 0.79_+0.05 nmol. The weights of the kidneys of the two groups were similar: 1.6_+0.2 and 1.5+-0.3 for the control and experimental groups, respectively. In addition, the kidney morphology of the rats treated as neonates with A V P appeared normal by light microscopic examination, DISCUSSION The kidneys of adult rats treated as neonates with AVP contain fewer binding sites for AVP than do those of control rats. The affinity of the AVP binding sites is unchanged, This decrease in kidney binding sites may be responsible for the previously reported insensitivy to AVP caused by neonatal administration of the peptide 151. Whether there is also an impairment in adenylate cyclase coupling of the receptors in these rats remains to be investigated,
40
60
80
100
PHI AVP BOUND, fmol/mg IXOtein FIG. 2. Scatchard plots of the saturation binding data.
Several hormones have been shown to regulate the concentration of their specific receptors in the adult. Manipulation of hormone levels to which target cells are exposed can either increase or decrease the number of receptors, without altering the affinity of the receptors (for reviews see [2,7]. AVP administered to adults can alter the responsiveness of the kidney to AVP, as measured by adenylate cyclase activation [9]. These changes are transient, however, and responsiveness returns to normal when hormone concentrations are restored to the steady-state level. In the present experiment, a brief neonatal exposure to high levels of AVP had a persistent influence on the number of kidney AVP receptors. The pituitary AVP content of these rats was similar to that of the control rats [Handelmann, unpublished data], suggesting that steady-state levels of AVP in the animal are not altered [61. The number of A V P binding sites is low in the rat kidney during the first week after birth, although binding sites are measurable and some stimulation of cAMP can be produced [8]. Other binding characteristics of the neonatal AVP receptor, such as binding affinity and specificity, are similar to those of the adult. There is a sharp increase in the number of sites between days 10 and 25 after birth. Premature exposure to high levels of AVP may therefore in some way interfere with the surge in receptor number in the second week after birth. These data support the hypothesis that neuropeptides influence the development of their receptors 141.
ACKNOWLEDGEMENTS The authors are grateful to Drs. H. Gainer and T. O'Donohue for helpful comments on this experiment and manuscript.
NEONATAL
AVP DECREASES
ADULT AVP BINDING
1219
REFERENCES 1. Bohlen, P., S. Stein, W. Dairman and S. Udenfriend. Fluorometric assay of proteins in the nanogram range. Arch Biochim Biophys 155: 213--220, 1973. 2. Catt. K. J.o J. P. Harwood, G. Aguilera and M. L. Dufau. Hormonal regulation of peptide receptor and target cell responses. Nature 280: 109-116, 1979. 3. Cole, B., D. Rodbard and P. Munson. Development of a friendly, self-teaching, interactive statistical package for the analysis of clinical research data: the BRIGHT stat-pack. Proc 7th Annu Syrup Comp Appl Med Care. 1983. 4. Handelmann, G. E. Neuropeptide administration to neonatal rats regulates sensitivity to peptides in adults. Neuroendocrinol Lett 5: 186, 1983. 5. Handelmann, G. E., J. T. Russell, H. Gainer, R. Zerbe and M. Bayorh. Vasopressin administration to neonatal rats reduces antidiuretic response in adult kidneys Peptides 4: 827-832, 1983.
6. Jones, C. W. and B. T. Picketing. 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, 1969. 7. Lesniak, M. A. and J. Roth. Regulation of receptor concentration by homologous hormones. J Biol Chem 251: 3720-3729, 1976. 8. Rajerison, R. M., D. Butlen and S. Jard. Ontogenetic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation. Mol Cell Endocrinol 4: 271-285, 1976. 9. Rajerison, R. M., D. Butlen and S. Jard. Effects of in vivo treatment with vasopressin and analogues on renal adenylate cyclase responsiveness to vasopressin stimulation in vitro. Endocrinology 101: 1-12, 1977.
0196-9781/84 $3.00 + .IX)
BRIEF C O M M U N I C A T I O N
Neonatal Exposure to Vasopressin Decreases Vasopressin Binding Sites in the Adult Kidney G A I L E. H A N D E L M A N N
z AND SAMUEL
C. S A Y S O N
Laboratory o f Developmental Neurobiology, N I C H D - N I H , Bethesda, M D 20205 R e c e i v e d 16 A u g u s t 1984 HANDELMANN, G. E. AND S. C. SAYSON. Neonatal exposure to vasopressin decreases vasopressin binding sites in the adult kidney. PEPTIDES 5(6) 1217-1219, 1984.mPrevious experiments showed that rats injected with vasopressin (AVP) during the first seven days after birth were less sensitive as adults to the antidiuretic effects of AVP than were control rats. In the present experiment, binding sites for AVP were measured in the kidneys of similarly treated adult rats. Neonatal exposure to AVP significantly decreased the number of binding sites in the adults, but did not affect the binding affinity of the sites. It is concluded that neonatal exposure to AVP which produces a long-lasting decrease in responsiveness of the kidney to AVP is correlated with a reduction in the number of AVP binding sites in the tissue. Vasopressin
Vasopressin binding sites
Kidney
Development
IN previous experiments, we showed that exogenously administered vasopressin (AVP), a neurohypophyseal peptide hormone, could influence the development o f one of its target organs, the kidney [5]. Administration of A V P to neonatal rats produced a long-term deleterious effect on the AVP-mediated osmoregulatory function of the kidney. The rats exhibited polyuria as adults, and their kidneys were relatively insensitive to A V P in a bioassay for antidiuresis. This insensitivity was correlated with a deficit in the kidney AVP-receptor mediated formation of cAMP, although parathyroid hormone- and caleitonin-stimulated c A M P formation in this tissue were normal. This deficit could therefore have been due to either a reduced number of A V P receptors, decreased binding efficiency of the receptors, or an impairment of the receptor-adenylate cyclase coupling. In the present experiment, we investigated AVP-reeeptor binding in the kidneys of adult rats treated as neonates with AVP, and we demonstrate that the number of binding sites is reduced in these rats.
buffered saline (PBS), pH 7.4, or A V P (4 ~g/100 g body weight, dissolved in PBS; Calbiochem, 395 U/rag). The assignment of pups within a litter to a treatment group was done at random. The pups were weaned on day 23.
Binding Assay When the rats were at least 90 days old, they were decapitated and their kidneys rapidly removed and frozen on powdered dry ice. The frozen kidneys were cut into 25 micron sections and the sections thaw-mounted on gelatin-coated slides. Sections taken through the largest aspect of the kidney, in which cortex, medulla, and collecting duets were clearly visible, were used for the assay. Adjacent sections were used for protein determination using fluorescamine [1] with bovine serum albumin as the standard. The slides were pre-incubated for 15 minutes at 25°C in 100 mM Tris pH 7.4, containing 0.2% bovine serum albumin, 10 mM MgCI~, 40 ~g/ml bacitracin, 6/~g/ml soybean trypsin inhibitor, and 0.1% lysine. The slides were removed from the preincubation buffer, and 200 p,l o f buffer containing varying amounts of aH-AVP (51 Ci/mmol; New England Nuclear) was placed directly onto the kidney section. The sections were incubated for 30 minutes at 25°C. At the end of the incubation, the slides were placed in racks and transferred sequentially through four one-minute rinses of 50 mM Tris, pH 7.4 at 4°C. Each kidney section was then wiped from the
METHOD
Subjects and Peptide Treatment Two litters of albino rat pups were cross-fostered and the litters culled to 10 pups each. Beginning the day after birth (neonatal day 11 and ending neonatal day 7. each pup received daily subcutaneous injections of either phosphate-
'Requests for reprints should be addressed to Dr. G. E. Handelmann, Lab. of Developmental Neurobiology, NICHD-NIH, Bldg. 36 Rm. 2A21. Bethesda, MD 20205. G.E.H. is a Pharmacology Research Associate of the National Institute of General Medical Sciences.
1217
1218
~
H A N D E L M A N N A N D SAYSON
70
~
I1 1
I 5
I 10
100
I I 20 30 PH] AVP, nmolm/lil
I 40
I 50
FIG. 1. Plot of specific binding of [:~H]AVPto kidney slices taken from adult rats treated as neonates with either AVP or PBS. *Significantly different from Neonatal Saline group (Student's t-test, p<0.05). Each point is the mean of duplicates from six rats,-+S.E.M.
• Neonatal AVP
~ 40
•
o
o
•
o
20 • e I
20
slide with a glass filter (Whatman OF/B), placed in 8 ml of Aquasol (New England Nuclear), vortexed, and the radioactivity counted. Total binding was determined by incubating sections with concentrations of aH-AVP ranging from 0.5 to 50 nM. Nonspecific binding was defined as the amount of radioactivity which remained bound when sections were incubated with 3H-AVP in the presence of 10-6 M unlabelled AVP. RESULTS
Binding Assay The Bma.~ and K,) values were obtained by regression analysis of Scatchard plots of saturation binding data (Fig. I), using a curve-fitting computer program (BRIGHT; [3]). The maximal binding capacity (Bma.O of kidney sections from rats treated as neonates with AVP was about 26% less than that of the control rats (Fig. 2), or 68__.6 and 92+_9 fmols/mg protein, respectively (p<0.05, Student's t-test). The Scatchard plots (Fig. 2) were linear, and indicated that the dissociation constants (K,) for the two groups' binding were similar, The K , for the group treated with AVP was 0.86+_0.06 nmol, and that for the control group was 0.79_+0.05 nmol. The weights of the kidneys of the two groups were similar: 1.6_+0.2 and 1.5+-0.3 for the control and experimental groups, respectively. In addition, the kidney morphology of the rats treated as neonates with A V P appeared normal by light microscopic examination, DISCUSSION The kidneys of adult rats treated as neonates with AVP contain fewer binding sites for AVP than do those of control rats. The affinity of the AVP binding sites is unchanged, This decrease in kidney binding sites may be responsible for the previously reported insensitivy to AVP caused by neonatal administration of the peptide 151. Whether there is also an impairment in adenylate cyclase coupling of the receptors in these rats remains to be investigated,
40
60
80
100
PHI AVP BOUND, fmol/mg IXOtein FIG. 2. Scatchard plots of the saturation binding data.
Several hormones have been shown to regulate the concentration of their specific receptors in the adult. Manipulation of hormone levels to which target cells are exposed can either increase or decrease the number of receptors, without altering the affinity of the receptors (for reviews see [2,7]. AVP administered to adults can alter the responsiveness of the kidney to AVP, as measured by adenylate cyclase activation [9]. These changes are transient, however, and responsiveness returns to normal when hormone concentrations are restored to the steady-state level. In the present experiment, a brief neonatal exposure to high levels of AVP had a persistent influence on the number of kidney AVP receptors. The pituitary AVP content of these rats was similar to that of the control rats [Handelmann, unpublished data], suggesting that steady-state levels of AVP in the animal are not altered [61. The number of A V P binding sites is low in the rat kidney during the first week after birth, although binding sites are measurable and some stimulation of cAMP can be produced [8]. Other binding characteristics of the neonatal AVP receptor, such as binding affinity and specificity, are similar to those of the adult. There is a sharp increase in the number of sites between days 10 and 25 after birth. Premature exposure to high levels of AVP may therefore in some way interfere with the surge in receptor number in the second week after birth. These data support the hypothesis that neuropeptides influence the development of their receptors 141.
ACKNOWLEDGEMENTS The authors are grateful to Drs. H. Gainer and T. O'Donohue for helpful comments on this experiment and manuscript.
NEONATAL
AVP DECREASES
ADULT AVP BINDING
1219
REFERENCES 1. Bohlen, P., S. Stein, W. Dairman and S. Udenfriend. Fluorometric assay of proteins in the nanogram range. Arch Biochim Biophys 155: 213--220, 1973. 2. Catt. K. J.o J. P. Harwood, G. Aguilera and M. L. Dufau. Hormonal regulation of peptide receptor and target cell responses. Nature 280: 109-116, 1979. 3. Cole, B., D. Rodbard and P. Munson. Development of a friendly, self-teaching, interactive statistical package for the analysis of clinical research data: the BRIGHT stat-pack. Proc 7th Annu Syrup Comp Appl Med Care. 1983. 4. Handelmann, G. E. Neuropeptide administration to neonatal rats regulates sensitivity to peptides in adults. Neuroendocrinol Lett 5: 186, 1983. 5. Handelmann, G. E., J. T. Russell, H. Gainer, R. Zerbe and M. Bayorh. Vasopressin administration to neonatal rats reduces antidiuretic response in adult kidneys Peptides 4: 827-832, 1983.
6. Jones, C. W. and B. T. Picketing. 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, 1969. 7. Lesniak, M. A. and J. Roth. Regulation of receptor concentration by homologous hormones. J Biol Chem 251: 3720-3729, 1976. 8. Rajerison, R. M., D. Butlen and S. Jard. Ontogenetic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation. Mol Cell Endocrinol 4: 271-285, 1976. 9. Rajerison, R. M., D. Butlen and S. Jard. Effects of in vivo treatment with vasopressin and analogues on renal adenylate cyclase responsiveness to vasopressin stimulation in vitro. Endocrinology 101: 1-12, 1977.