Original Paper
Pharmacology 1992;44:245-256
a Department of Pharmacology, School of Medicine. East Carolina University, Greenville, N.C., b Neuropharmacology Section, Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, N.C., USA
Age-Related Changes in Opioid Peptide Concentrations in Brain and Pituitary of Spontaneously Hypertensive Rats Effect of Antihypertensive Drugs and Comparison with Deoxycorticosterone Acetate and Salt Hypertension
Keyw ords
Abstract
Antihypertensive drugs Deoxycorticosterone acetate + salt hypertension Dynorphins Endorphins Enkephalins Hypertension Neural control of circulation Opioid peptides Spontaneously hypertensive rat
The relationship of age-dependent changes in concentrations of various opioid peptides in the brain and pituitary to the development of hyperten sion was studied in the spontaneously hypertensive rat (SHR). Normotensive Wistar-Kyoto (WKY) and Sprague-Dawley rats served as controls. Opioids determined were dynorphin A (1-8) [DN-A(l-8)], p-endorphin (BE) and Met-enkephalin (ME). Three approaches were used: (1) temporal correlations of opioid concentrations with the onset of hypertension in 4-, 8-, 12- and 16-week-old rats; (2) study of opioid changes when hyperten sion development was prevented with antihypertensive drugs and (3) de termination of possible opioid peptide changes in another rat model of hypertension, the deoxycorticosterone acetate (DOCA) + salt model. Opioid peptide concentration differences (SHR/ WKY) found were as fol lows. There were much lower DN-A(l-8) levels in the SHR hippocampus and hypothalamus at all ages studied. At 12 and 16 weeks, coincidently with the onset of hypertension, lower levels of BE were found in the ante rior lobe of the pituitary, but there were higher BE and ME levels found in the neurointermediate lobe (NIL). Prevention of hypertension in SHR by 8 weeks of oral therapy with guanethidine and hydralazine reversed the BE and ME changes in the NIL but not in the anterior lobe. There were no brain or pituitary changes in opioid peptide concentrations associated with DOCA-salt hypertension. The results are interpreted as supporting a role for altered concentrations of brain and pituitary opioids in the genesis of SHR hypertension.
Received: July 12,1991 Accepted: September 10,1991
Dr. Alphonse J. Ingenito Department of Pharmacology School of Medicine Greenville, NC 27858-4354 (USA)
©1992 S. Karger AG, Basel 0031-7012/92/ 044 5-0245S2.75/0
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 8:14:19 AM
Si-Jia L ia So C. Wonga Jau-Shyong Hongb Alphonse J. Ingenitoa
Endogenous opioid peptides appear to have important roles as both central and pe ripheral neuromodulators in the neural and endocrine control of cardiovascular function [1]. Evidence supporting such roles include ( 1) the profound cardiovascular effects which these agents cause on systemic administration or local injection into the brain [1,2], (2) the anatomical location of these peptides in car diovascular control areas of the brain and in conjunction with peripheral autonomic nerves [3, 4] and (3) the ability of naloxone and other opiate receptor antagonists to atten uate cardiovascular effects said to be associ ated with endogenous opioids, e.g. hemody namic shock, spinal injury and stroke [5, 6]. Considerable suggestive evidence of an opioid-cardiovascular relationship in hyper tension comes from studies on the differential cardiovascular effects of opioids and opioid blocking drugs between the spontaneously hy pertensive rat (SHR) and normotensive con trols [7-9], Further support for an opioidhypertension relationship in SHR arises from reports on differences in concentrations of various opioid peptides in various areas of SHR brain compared with normotensive con trols [ 10-22], Additionally, there appear to be differences in preponderance of opioid recep tor subtypes in SHR brain compared to Wistar-Kyoto (WKY) controls, from q to k [23] or p to 5 [8]. While the reported differences in concentrations of opioid peptides or opioid receptor subtypes in various brain areas be tween SHR and WKY rates are suggestive of a possible causal relationship of these differ ences to SHR hypertension, definitive evi dence of causality is lacking. Accordingly, the purpose of the present investigation was to acquire evidence for or against a causal relationship between brain and pituitary opioid concentration changes
246
and development of hypertension in SHR. The approaches used were: (1) estimation of opioid concentrations at different stages (ages) during hypertension development, (2) estimation of brain opioids following pre vention of hypertension development in SHR by chronic administration of antihyperten sive drugs and (3) determination of opioid peptide concentrations in brain and pituitary in a nongenetic form of hypertension in the rat, the deoxycorticosterone acetate (DOCA) + salt model.
Materials and Methods Experimental A nirnals
Healthy, male SHR. their genetically matched nor motensive WKY controls and normotensive SpragueDawley (SD) rats, obtained from Charles River Labo ratories, ages 4, 8. 12 and 16 weeks, were used in this study. They were housed in an animal facility, 3-4 to a cage, at a constant temperature of 23 °C. with 12 h light/12 h dark cycles, and were fed standard rat chow and tap water ad libitum. The animals were brought into the facility 1 week before use in the study. The work was done in 4 separate phases, with rats in each phase being acquired from the supplier at different times. In the first phase, only 16-week-old SHR. WKY and SD rats were used, and a variety of brain areas was analyzed for opioids. In the second phase only pitu itary opioids were determined in SHR and WKY rats of ages 4, 8, 12 and 16 weeks. In the third phase pitu itary opioid peptides were studied in SHR and WKY in which hypertension was prevented using antihyper tensive drugs (see below). In the fourth phase, pituitary opioids were determined in SD rats made hypertensive by unilateral nephrectomy and administration of DOCA and salt (see below). Blood Pressure and Weight Determinations
Arterial systolic pressures were determined in rat tail arteries with an IITC tail cuff apparatus. The pro cedure requires brief (no longer than 5 min) immobili zation of rats in Plexiglas holders and a slight elevation of room temperature to 27 °C. To reduce the influence of immobilization stress on these determinations, they were done on 3 consecutive days, just prior to sacrifice for opioid peptide determinations, with data only from the last determination day being reported here. Read
U/Wong/Hong/Ingenito
Brain Opioids in Hypertension
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 8:14:19 AM
Introduction
Antihypertensive Drug Therapy
One group of 10 each WKY rats and two groups of 10 each SHR at 8 weeks of age were given drinking water as follows: (l)WKY. tap water: (2)SHR. tap water: (3) SHR. tap water with antihypertensive drugs (hydralazine chloride and guanethidine monosulfate, 25 mg/kg/24 h for each drug; Ciba-Geigy). Animal body weight and the average total amount of drinking water consumed in 24 h per rat were determined every week. Using these figures the concentration of drugs in drinking water was adjusted weekly to deliver the stated doses. Administration continued for 8 weeks. The arterial pressure of each rat was determined before and during therapy once a week. Preparation ofDOCA-Salt-Hypertensive Rats
Five-week-old SD rats were anesthetized with keta mine (117 mg/kg. s.c.) and xylazine (13 mg/kg. s.c.). The left kidney was surgically removed from each ani mal. One week after surgery the rats were divided into two groups (n = 10 for each). One group was treated with DOCA (Sigma, 40 mg/kg, s.c., suspended in sesame oil. once a week for 5 weeks) and made to drink high-salt water (8.0% NaCl in tap water). The control group was given injections of sesame oil (0.5 ml. s.c,, once a week) and tap water to drink. Blood pressure was determined once a week before and after treat ment. Opioid peptides were then determined in the pituitaries of these II-week-old rats, whose body weights were similar to those of 16-week SHR and WKY. SD rats have a more accelerated growth curve than SHR and WKY rats. We felt it more appropriate to use weight-matched, rather than age-matched rats for the comparison of opioid peptide changes in brain between genetic and DOCA-salt hypertension. SD rather than WKY rats were selected for the DOCA-salt treatment because we wished to study a model of hypertension devoid of any possibility of a genetic form of hypertension, such as might have been possi ble with a DOCA-salt/WKY combination. Preparation o f Tissue
Two days following the last determinaton day of blood pressure in any particular age group, the animals were sacrificed by decapitation in the morning. Brains
and pituitaries were rapidly removed, and the areas to be studied were dissected immediately on an icechilled plastic plate, then frozen on dry ice and stored at -7 0 °C until extraction. On the day of extraction each separate frozen tissue was homogenized in 1.0 ml of 2 A' acetic acid and immersed in boiling water for 5 min. After centrifugation, the supernatants were iyophilized, and the residues were reconstituted in I ml of phosphate buffer (0.02 mol/1. pH 7.4). After homog enization and centrifugation again, aliquots of from 0.01 to 150 pi (depending on the particular opioid and brain tissue) of supernatant of each brain sample was used for radioimmunoassay. Radioimmunoassay
Tissue levels of opioid peptides were determined by a radioimmunoassay method. The antibodies re spectively against (3-endorphin (BE). Met-enkephalin (ME) and dynorphin A (1-8) [DN-A(l-8)] were pre pared by J.S.H. All peptides were purchased from Sigma Chemical Company. The radiolabeled peptides. I25I-BE and l25I-ME were purchased from Amcrsham Company: l25l-DN-A( 1-8) was prepared by Hazleton Biotechnological Company. Antiserum against BE did not cross-react with adrenocorticotropic hormone. ME, Leu-enkephalin. DN-A( 1-8) and ME-Arg-Phe but cross-reacted with [3lipotropin to the extent of 60%. Antiserum against ME had very low cross-reactivities with DN-A(l-8) (< 0.06%), BE (
Pharmacology 1992;44:245-256
a Department of Pharmacology, School of Medicine. East Carolina University, Greenville, N.C., b Neuropharmacology Section, Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, N.C., USA
Age-Related Changes in Opioid Peptide Concentrations in Brain and Pituitary of Spontaneously Hypertensive Rats Effect of Antihypertensive Drugs and Comparison with Deoxycorticosterone Acetate and Salt Hypertension
Keyw ords
Abstract
Antihypertensive drugs Deoxycorticosterone acetate + salt hypertension Dynorphins Endorphins Enkephalins Hypertension Neural control of circulation Opioid peptides Spontaneously hypertensive rat
The relationship of age-dependent changes in concentrations of various opioid peptides in the brain and pituitary to the development of hyperten sion was studied in the spontaneously hypertensive rat (SHR). Normotensive Wistar-Kyoto (WKY) and Sprague-Dawley rats served as controls. Opioids determined were dynorphin A (1-8) [DN-A(l-8)], p-endorphin (BE) and Met-enkephalin (ME). Three approaches were used: (1) temporal correlations of opioid concentrations with the onset of hypertension in 4-, 8-, 12- and 16-week-old rats; (2) study of opioid changes when hyperten sion development was prevented with antihypertensive drugs and (3) de termination of possible opioid peptide changes in another rat model of hypertension, the deoxycorticosterone acetate (DOCA) + salt model. Opioid peptide concentration differences (SHR/ WKY) found were as fol lows. There were much lower DN-A(l-8) levels in the SHR hippocampus and hypothalamus at all ages studied. At 12 and 16 weeks, coincidently with the onset of hypertension, lower levels of BE were found in the ante rior lobe of the pituitary, but there were higher BE and ME levels found in the neurointermediate lobe (NIL). Prevention of hypertension in SHR by 8 weeks of oral therapy with guanethidine and hydralazine reversed the BE and ME changes in the NIL but not in the anterior lobe. There were no brain or pituitary changes in opioid peptide concentrations associated with DOCA-salt hypertension. The results are interpreted as supporting a role for altered concentrations of brain and pituitary opioids in the genesis of SHR hypertension.
Received: July 12,1991 Accepted: September 10,1991
Dr. Alphonse J. Ingenito Department of Pharmacology School of Medicine Greenville, NC 27858-4354 (USA)
©1992 S. Karger AG, Basel 0031-7012/92/ 044 5-0245S2.75/0
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 8:14:19 AM
Si-Jia L ia So C. Wonga Jau-Shyong Hongb Alphonse J. Ingenitoa
Endogenous opioid peptides appear to have important roles as both central and pe ripheral neuromodulators in the neural and endocrine control of cardiovascular function [1]. Evidence supporting such roles include ( 1) the profound cardiovascular effects which these agents cause on systemic administration or local injection into the brain [1,2], (2) the anatomical location of these peptides in car diovascular control areas of the brain and in conjunction with peripheral autonomic nerves [3, 4] and (3) the ability of naloxone and other opiate receptor antagonists to atten uate cardiovascular effects said to be associ ated with endogenous opioids, e.g. hemody namic shock, spinal injury and stroke [5, 6]. Considerable suggestive evidence of an opioid-cardiovascular relationship in hyper tension comes from studies on the differential cardiovascular effects of opioids and opioid blocking drugs between the spontaneously hy pertensive rat (SHR) and normotensive con trols [7-9], Further support for an opioidhypertension relationship in SHR arises from reports on differences in concentrations of various opioid peptides in various areas of SHR brain compared with normotensive con trols [ 10-22], Additionally, there appear to be differences in preponderance of opioid recep tor subtypes in SHR brain compared to Wistar-Kyoto (WKY) controls, from q to k [23] or p to 5 [8]. While the reported differences in concentrations of opioid peptides or opioid receptor subtypes in various brain areas be tween SHR and WKY rates are suggestive of a possible causal relationship of these differ ences to SHR hypertension, definitive evi dence of causality is lacking. Accordingly, the purpose of the present investigation was to acquire evidence for or against a causal relationship between brain and pituitary opioid concentration changes
246
and development of hypertension in SHR. The approaches used were: (1) estimation of opioid concentrations at different stages (ages) during hypertension development, (2) estimation of brain opioids following pre vention of hypertension development in SHR by chronic administration of antihyperten sive drugs and (3) determination of opioid peptide concentrations in brain and pituitary in a nongenetic form of hypertension in the rat, the deoxycorticosterone acetate (DOCA) + salt model.
Materials and Methods Experimental A nirnals
Healthy, male SHR. their genetically matched nor motensive WKY controls and normotensive SpragueDawley (SD) rats, obtained from Charles River Labo ratories, ages 4, 8. 12 and 16 weeks, were used in this study. They were housed in an animal facility, 3-4 to a cage, at a constant temperature of 23 °C. with 12 h light/12 h dark cycles, and were fed standard rat chow and tap water ad libitum. The animals were brought into the facility 1 week before use in the study. The work was done in 4 separate phases, with rats in each phase being acquired from the supplier at different times. In the first phase, only 16-week-old SHR. WKY and SD rats were used, and a variety of brain areas was analyzed for opioids. In the second phase only pitu itary opioids were determined in SHR and WKY rats of ages 4, 8, 12 and 16 weeks. In the third phase pitu itary opioid peptides were studied in SHR and WKY in which hypertension was prevented using antihyper tensive drugs (see below). In the fourth phase, pituitary opioids were determined in SD rats made hypertensive by unilateral nephrectomy and administration of DOCA and salt (see below). Blood Pressure and Weight Determinations
Arterial systolic pressures were determined in rat tail arteries with an IITC tail cuff apparatus. The pro cedure requires brief (no longer than 5 min) immobili zation of rats in Plexiglas holders and a slight elevation of room temperature to 27 °C. To reduce the influence of immobilization stress on these determinations, they were done on 3 consecutive days, just prior to sacrifice for opioid peptide determinations, with data only from the last determination day being reported here. Read
U/Wong/Hong/Ingenito
Brain Opioids in Hypertension
Downloaded by: Nagoya University 133.6.82.173 - 1/13/2019 8:14:19 AM
Introduction
Antihypertensive Drug Therapy
One group of 10 each WKY rats and two groups of 10 each SHR at 8 weeks of age were given drinking water as follows: (l)WKY. tap water: (2)SHR. tap water: (3) SHR. tap water with antihypertensive drugs (hydralazine chloride and guanethidine monosulfate, 25 mg/kg/24 h for each drug; Ciba-Geigy). Animal body weight and the average total amount of drinking water consumed in 24 h per rat were determined every week. Using these figures the concentration of drugs in drinking water was adjusted weekly to deliver the stated doses. Administration continued for 8 weeks. The arterial pressure of each rat was determined before and during therapy once a week. Preparation ofDOCA-Salt-Hypertensive Rats
Five-week-old SD rats were anesthetized with keta mine (117 mg/kg. s.c.) and xylazine (13 mg/kg. s.c.). The left kidney was surgically removed from each ani mal. One week after surgery the rats were divided into two groups (n = 10 for each). One group was treated with DOCA (Sigma, 40 mg/kg, s.c., suspended in sesame oil. once a week for 5 weeks) and made to drink high-salt water (8.0% NaCl in tap water). The control group was given injections of sesame oil (0.5 ml. s.c,, once a week) and tap water to drink. Blood pressure was determined once a week before and after treat ment. Opioid peptides were then determined in the pituitaries of these II-week-old rats, whose body weights were similar to those of 16-week SHR and WKY. SD rats have a more accelerated growth curve than SHR and WKY rats. We felt it more appropriate to use weight-matched, rather than age-matched rats for the comparison of opioid peptide changes in brain between genetic and DOCA-salt hypertension. SD rather than WKY rats were selected for the DOCA-salt treatment because we wished to study a model of hypertension devoid of any possibility of a genetic form of hypertension, such as might have been possi ble with a DOCA-salt/WKY combination. Preparation o f Tissue
Two days following the last determinaton day of blood pressure in any particular age group, the animals were sacrificed by decapitation in the morning. Brains
and pituitaries were rapidly removed, and the areas to be studied were dissected immediately on an icechilled plastic plate, then frozen on dry ice and stored at -7 0 °C until extraction. On the day of extraction each separate frozen tissue was homogenized in 1.0 ml of 2 A' acetic acid and immersed in boiling water for 5 min. After centrifugation, the supernatants were iyophilized, and the residues were reconstituted in I ml of phosphate buffer (0.02 mol/1. pH 7.4). After homog enization and centrifugation again, aliquots of from 0.01 to 150 pi (depending on the particular opioid and brain tissue) of supernatant of each brain sample was used for radioimmunoassay. Radioimmunoassay
Tissue levels of opioid peptides were determined by a radioimmunoassay method. The antibodies re spectively against (3-endorphin (BE). Met-enkephalin (ME) and dynorphin A (1-8) [DN-A(l-8)] were pre pared by J.S.H. All peptides were purchased from Sigma Chemical Company. The radiolabeled peptides. I25I-BE and l25I-ME were purchased from Amcrsham Company: l25l-DN-A( 1-8) was prepared by Hazleton Biotechnological Company. Antiserum against BE did not cross-react with adrenocorticotropic hormone. ME, Leu-enkephalin. DN-A( 1-8) and ME-Arg-Phe but cross-reacted with [3lipotropin to the extent of 60%. Antiserum against ME had very low cross-reactivities with DN-A(l-8) (< 0.06%), BE (
