Down-regulation of alpha 2 adrenoceptors in ventrolateral medulla of spontaneously hypertensive rats.

The binding of [3H]idazoxan [correction of idaxazon] to imidazole sites and [3H]rauwolscine to alpha 2 adrenoceptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]idazoxan [correction of idaxazon] bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [3H]idazoxan [correction of idaxazon] in ventrolateral medulla and cerebral cortex was found to be similar in SHR and WKY rats. [3H]Rauwolscine bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [3H]rauwolscine in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [3H]rauwolscine binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due a decrease (32%) in the Bmax value in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. It is concluded that imidazole binding sites are not affected while, alpha 2 adrenergic binding sites are decreased in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.     

Characteristics of central binding sites for [3H] DAMGO in spontaneously hypertensive rats

The binding of [3H] DAMGO, a highly selective ligand for mu-opiate receptors, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. [3H] DAMGO bound to membranes of brain regions and spinal cord at a single high affinity site. The receptor density (Bmax value) and apparent dissociation constant (Kd value) of [3H] DAMGO to bind to membranes of hippocampus, corpus striatum, pons and medulla, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of [3H] DAMGO in membranes of hypothalamus and midbrain of SHR rats was significantly higher than in WKY rats but the Kd values in the two strains did not differ. On the other hand, the Bmax value of [3H] DAMGO in membranes of amygdala of SHR rats was lower than that of WKY rats but the Kd values in the two strains were similar. It is concluded that SHR rats have higher density of mu-opiate receptors in hypothalamus and midbrain but lower density in amygdala in comparison with WKY rats, and that such differences in the distribution of mu-opiate receptors may be related to the elevated blood pressure in SHR rats.     

Down-regulation of endothelin receptors in the ventrolateral medulla of spontaneously hypertensive rats.

The binding of [125I] sarafotoxin 6b (SRT 6b) and [125I] endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [125I] SRT 6b bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [125I] SRT 6b in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [125I] SRT 6b binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to decrease (48%) in the Bmax values in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. [125I] ET-1 also bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [125I] ET-1 in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [125I] ET-1 binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to 36% decrease in the Bmax values in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. It is concluded that the population of ET receptors is less in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

Changes in renal, platelet and cardiac nitrobenzylthioinosine binding in spontaneously hypertensive rats

In an attempt to investigate the role of nucleoside transporter function in the hypertensive state, we have compared the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR), a nucleoside transporter probe, in membranes prepared from platelet, renal, pulmonary, cardiac and brain tissues of spontaneously hypertensive rats (SHR) to those of age-matched Wistar-Kyoto (WKY) controls. At 4 weeks of age, [( 3H]NBMPR) binding sites (Bmax) increased in the kidney of SHR but decreased in platelets, whereas no changes were found in the heart, lung or brain. At 18 weeks of age, [3H]NBMPR binding sites (Bmax) remained increased in the kidney and decreased in platelets with no changes in the other tissues. The only change in apparent binding affinity (KD) was an increase in the heart of SHR at 4 weeks. Age-dependent decreases were also observed in the heart and platelets of both SHR and WKY at 18 weeks. The results indicate that the changes in binding characteristics may be due to a combination of the pharmacodynamic differences between the strains, age, as well as to the pathogenesis of hypertension. Consequently, it cannot be concluded that the altered binding characteristics are the result of the elevated blood pressure.     

Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats

The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound specifically to rat brain membranes at a single high affinity site. The density (Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR rats compared to WKY rats. The apparent dissociation constants (Kd values) for the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the various brain regions revealed that the density of [3H]MeTRH was highest in the hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex, hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To assess the significance of increased binding sites for [3H]MeTRH in some brain regions of SHR rats, the binding studies were carried out during normotensive and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old SHR rats there was neither an increase in blood pressure nor any increase in [3H]MeTRH binding in the hypothalamus and striatum as compared to age matched WKY rats. With the development of elevated blood pressure at 6 weeks, an increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in comparison to the tissues from WKY rats was observed. The results provide, for the first time, evidence for a parallel increase in the density of brain TRH receptors with elevation of blood pressure, and suggest that brain TRH receptors may play an important role in the pathophysiology of hypertension.     

Atrial natriuretic peptide and angiotensin II binding sites in cerebral capillaries of spontaneously hypertensive rats

1. We carried out investigations on specific atrial natriuretic peptide (ANP) and angiotensin II (ANG) binding sites in capillaries isolated from the cerebral cortex of spontaneously hypertensive rats (SHR), an animal model of human essential hypertension, and also from Wistar Kyoto rats (WKY). 2. In an equilibrium binding study done in the presence of increasing concentrations of the radiolabeled ligands, the binding of 125I-rat alpha-ANP (1-28) [ANF-(99-126)] (125I-rANP) and 125I-ANG (5-L-isoleucine) (125I-ANG) to the cerebral capillaries was single and of a high affinity. 3. The maximum binding capacity (Bmax) and dissociation constant (Kd) in the 125I-rANP binding of 20-week-old, hypertensive SHR was significantly lower than in age-matched, normotensive WKY. Conversely, a significant increase in the Bmax of 125I-ANG binding of adult SHR was observed, with a significant decrease in the Kd. 4. There was no differences in the Bmax of 125I-rANP and 125I-ANG binding between 4-week-old, prehypertensive SHR and age-matched WKY. However, there was a significant decrease in the Kd of 125I-rANP binding of SHR. 5. As a dramatic change in the binding kinetics of 125I-rANP and 125I-ANG was noted in the cerebral capillaries of adult sustained-hypertensive SHR, the possibility that ANP and ANG play a role in the etiology of dysfunction of the blood-brain barrier complicated with hypertension, by interacting with specific receptors, would have to be considered.     

Alterations in a hypothalamic GABA system in the spontaneously hypertensive rat

The density (Bmax) of muscimol and clonazepam binding to hypothalamic membranes from spontaneously hypertensive rats (SHR) was reduced compared to age-matched Wistar Kyoto (WKY) animals in the period 80 – 120 days. There were no significant differences in dissociation constant (Kd) for either ligand at this time. At 30 – 36 days, prior to development of pronounced hypertension, there were no differences in Kd or Bmax for either ligand in SHR and WKY animals. There were also deficits in endogenous hypothalamic GABA concentrations in SHR at 75 and 120 days as compared to WKY. The hypothesis is advanced, that there may be a dysfunction of a hypothalamic GABA system in the SHR rat as hypertension develops.     

Pregnancy related changes of opiate receptors identified in rat uterine membranes by 3H-naloxone binding

3H-Naloxone was used to demonstrate the presence of specific opiate binding sites in uterine membrane preparations of rats. 3H-Naloxone binding (0.41-27 nM) was found to be rapid, saturable and reversible showing two populations of binding sites with the characteristic of high (KD 2.2 nM; Bmax 46.6 fmol/mg prot.) and low (KD 18.1 nM; Bmax 143.7 fmol/mg prot.) affinity. The number and affinity of the binding sites labelled by 3H-naloxone in the uterus were measured in the rat at mid (14 days), late (21 days) pregnancy and at parturition. The high and low affinity recognition sites labelled by 3H-naloxone showed a consistent reduction during pregnancy and at parturition without changes in the affinity constant. We concluded that pregnancy and parturition are associated with significant changes in the number of the opiate receptors bound in the uterus by 3H-naloxone. This phenomenon which seems to be linked with the several pregnancy-related changes in the levels of endogenous peptides and hormones could be relevant to further explain the pregnancy related changes in pain perception and maternal behavior.     

Effects of paradoxical sleep deprivation on the activity of opiate receptors isolated from synaptic membranes of the rat brain]

The specific binding of 3H-naloxone with opiate receptors isolated from brain synaptic membranes of control and paradoxical sleep deprived rats were studied. This extreme state was shown to reduce the naloxone-binding activity of synaptic membranes by 35% and isolated receptors by 25-28%. The values of Kd and Bmax were calculated for isolated opiate receptors at different stages of purification. Considerable decrease of 3H-naloxone binding sites density (2 times) in the isolated opiate receptors with simultaneous increase of their affinity (3-4,5 times) was found following 24 hours paradoxical sleep deprivation. These findings suggest development of fixed alterations in the structure and functions of integral receptor proteins under extreme influences.     

Effects of subacute treatment with cocaine on activities of N-demethylase, UDP-glucuronyltransferase and sulfotransferase in WKY and SHR rat liver--sex and strain differences

The effects of subacute treatment with cocaine on activities of cocaine N-demethylase, UDP-glucuronyltransferase (GT) toward 4-nitrophenol and phenolphthalein and sulfotransferase (ST) toward androsterone and 4-nitrophenol in livers from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were investigated. Hepatic metabolism of cocaine was different between the sexes (with males having higher N-demethylase activity) and the strains (with WKY rats having higher activity). The effects of subacute cocaine administration on the activity of cocaine N-demethylase were also sex- and strain-related. Whereas cocaine administration increased activity of hepatic N-demethylase in both female strains, it decreased activity in male WKY and had no effect on activity in male SHR. Sex and strain-related as well as cocaine-induced differences were also found in activities of hepatic GT toward 4-nitrophenol and phenolphthalein as well as in activity of hepatic ST towards andersterone and 4-nitrophenol. These results suggest that some of the individual variation in the effects of cocaine may be due to sex and genetic differences in the hepatic metabolism of cocaine and/or in sexually and/or/genetically-determined differences in how cocaine affects hepatic metabolism of other xenobiotics.     

Differential changes in atrial natriuretic peptide and vasopressin receptor bindings in kidney of spontaneously hypertensive rat

To elucidate the role of atrial natriuretic peptide (ANP) and vasopressin (VP) in a hypertensive state, ANP and VP receptor bindings in spontaneously hypertensive rat (SHR) kidney were analyzed using the radiolabeled receptor assay (RRA) technique. Systolic blood pressure of SHR aged 12 weeks was statistically higher than that of age-matched Wistar Kyoto (WKY) rats. Maximum binding capacity (Bmax) of [125I]-ANP binding to the SHR kidney membrane preparations was statistically lower than that of WKY rats, but dissociation constant (Kd) was not significantly different. On the other hand, Bmax of [3H]-VP binding to the SHR kidney membrane preparations was statistically higher than that of WKY rats, but Kd were similar. Since the physiological action of ANP is natriuresis and VP is the most important antidiuretic hormone in mammalia, these opposite changes of ANP and VP receptor bindings in SHR kidney suggested that these peptides may play an important role in the pathophysiology of the hypertensive state, although it has not been confirmed as yet.     

Decreased angiotensin II binding affinity and binding capacity in the anterior pituitary gland of adult spontaneously hypertensive rats

Angiotensin II (ANG) binding sites were quantified in single pituitary glands from 4-week-old and 14-week-old male spontaneously hypertensive rats (SHR) and age-matched male normotensive Wistar-Kyoto (WKY) control rats after incubation with 125I-[Sar1]-ANG, autoradiography with computerized densitometry, and comparison to 125I-standards. The maximum binding capacity (Bmax) decreased while the dissociation constant (Kd) for ANG increased in 14-week-old SHR when compared to age-matched WKY control rats (Bmax: 265 +/- 9 and 224 +/- 4 fmol/mg protein; Kd: 0.79 +/- 0.04 and 1.14 +/- 0.08 10(-9) M in WKY and SHR, respectively). Conversely, no difference between rat strains was found in 4-week-old animals. Our results suggest that pituitary ANG binding sites may play a role in the pathophysiology of established genetic hypertension.     

Effects of acute and subacute cocaine administration on the CNS dopaminergic system in Wistar-Kyoto and spontaneously hypertensive rats: II. Dopamine receptors

The characteristics of D-1 and D-2 dopamine receptors after acute and subacute cocaine administration were determined in striata and nuclei accumbens from WKY and SHR. In striata from acutely treated rats, significant increases in D-2 receptor density were observed at 30 min, 2 or 24 h following cocaine injection in both strains without changes in affinities. The density of D-1 receptors was significantly decreased 30 min after the injection in WKY, but not in SHR. In striata from subacutely treated rats, the density of D-1 receptors was significantly increased in 3- and 7-day treated WKY, but not in SHR. The affinities of both binding sites remained unchanged. In nuclei accumbens, the changes in both D-1 and D-2 receptors after cocaine administration were similar to those observed in the striatum. The results suggest that cocaine administration alters dopamine receptor binding characteristics. Furthermore, D-1 and D-2 dopamine receptors appear to be differently regulated.     

Binding sites for atrial natriuretic factor (ANF) in kidneys and adrenal glands of spontaneously hypertensive (SHR) rats

Binding sites for atrial natriuretic factor (ANF) were studied in kidneys and adrenal glands of 17 week old male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats by quantitative autoradiography using 125I-ANF-28. In kidney, 125I-ANF-28 binding sites were found in high concentrations in glomeruli and in much lower concentrations in the renal papilla. In adrenal gland, 125I-ANF-28 binding sites were highly localized to the zona glomerulosa and were of moderate density in the inner cortical regions. ANF binding sites did not occur in the adrenal medulla. The maximum binding capacity (Bmax) of 125I-ANF-28 was reduced by 50% in the kidney glomeruli of SHRs compared to WKY controls. In contrast, the affinity constant (Ka) for 125I-ANF-28 was elevated by 100% in kidney glomeruli of SHRs. There were no significant strain differences in values for Bmax or Ka for 125I-ANF-28 binding in the adrenal zona glomerulosa. These findings suggest that the natriuretic and diuretic actions of ANF within kidney glomeruli may be compromised in adult SHR rats and these alterations may contribute to the development and maintenance of hypertension in rats of this strain.     

Differential sensitivity to cocaine in spontaneously hypertensive and Wistar-Kyoto rats

Physiological, pharmacological and toxicological responses to two regimens of cocaine administration were compared between spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. An initial experiment examined renal excretory and hemodynamic function in response to an acute volume load in anesthetized SHR and WKY following subacute cocaine treatment (20 mg/kg, s.c., twice a day for 9 days). Anticipated renal responses to volume loading were obtained but the responses of cocaine-treated SHR and WKY did not differ from vehicle-treated rats. A second group of experiments compared responses to continuous i.v. infusions of cocaine (1.25 mg/kg.min). In freely moving animals, no differences were noted between SHR and WKY in the increases in mean blood pressure (MBP) and heart rate (HR) produced during cocaine infusion. The elapsed time-to-onset of convulsions (Tc) elicited by cocaine was similar in both strains. However, when rats were subjected to restraint during the infusion period, pressor and tachycardic responses were observed to be significantly less in WKY than in SHR or in freely moving rats of either strain. Restraint also differentially affected rectal temperature (RT) responses to cocaine. Hypothermic responses to cocaine were observed in all WKY. Both hypothermic and hyperthermic responses were observed in SHR. A significant correlation was demonstrated between the Tc and the maximal change in RT produced during cocaine infusion. Division of SHR into two arbitrary groups was made, based on the direction of cocaine-induced change in RT. A significant (p less than 0.01) shortening of the Tc was obvious in SHR (8 of 15) in whom cocaine produced a hyperthermia. These animals were designated SHRH. The mean value for Tc in those SHR which demonstrated a lowering in RT (SHRL; 7 of 15) in response to cocaine was similar to that for WKY. Moreover, the SHRH evidenced significantly greater increases in HR, but not MBP, to cocaine infusion than did SHRL. The results indicate that restraint stress causes expression of a significant heterogeneity in the RT response of SHR to cocaine. The magnitude and direction of the RT responses are negatively correlated with sensitivity to the convulsive effects of cocaine in SHR. Stress may modify toxic responses to cocaine by interactions with body temperature homeostasis.     

Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). However, sensitivity to Gpp[NH]p, isoproterenol plus GTP or Gpp[NH]p was significantly greater in the SHR. Beta-receptor density measured by [3H]DHA binding was unaltered. The dissociation constant, Kd, revealed a significantly greater binding affinity of the radioligand in the SHR (6.23 +/- 0.45 nM) compared with the WKY (8.53 +/- 0.82 nM). Activity of Gs was assessed by complementing S49 cyc- acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp[NH]p, and isoproterenol plus Gpp[NH]p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary Gs in the hypertensive rats.     

Inhibitory effect of Ca(2+) on ATP-mediated stimulation of NPR-A-coupled guanylyl cyclase in renal glomeruli from spontaneously hypertensive and normotensive rats.

Atrial natriuretic peptide (ANP) regulates blood pressure mainly through the occupation of the guanylyl cyclase-coupled receptor NPR-A, which requires ATP interaction for maximal activation. This study investigates the effect of extracellular Ca(2+) on ATP-mediated regulation of NPR-A-coupled guanylyl cyclase activity in glomerular membranes from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). ATP induced a significant increase in basal and ANP(1-28)-stimulated guanylyl cyclase activity that was greater in SHR than in WKY. Extracellular Ca(2+) inhibited ATP-stimulated guanylyl cyclase activity in a concentration-dependent manner, but did not modify basal and ANP(1-28)-stimulated guanylyl cyclase activity. In the presence of ATP, NPR-A showed higher affinity for ANP(1-28) and lower Bmax. Ca(2+) did not modify NPR-A-ANP(1-28) binding properties. The different effects of extracellular Ca(2+) on ANP(1-28)- or ATP-mediated guanylyl cyclase activation suggest that these events are differentially regulated. Addition of extracellular Ca(2+) induced similar effects in hypertensive and normotensive rats, suggesting that it is not responsible for the elevated cGMP production observed in SHR.     

A-type natriuretic peptide receptor in the spontaneously hypertensive rat kidney

Renal NPR-A binding characteristics was examined in SHR. Renal ANP binding sites of NPR-A showed a lower maximal binding capacity and higher affinity in SHR than in WKY at all intrarenal sites. Despite the lower B(max) in SHR, both ANP(1-28) and ANP(5-25) stimulate similar or greater cGMP production in isolated glomeruli. Studies on guanylate cyclase from glomerular and papillary membranes have reported an increased basal and stimulated guanylate cyclase activity in SHR. The present study provides further evidences for altered NPR-A receptors in SHR kidney, which might act as a negative feedback in response to hypertension.     

Effects of acute and subacute cocaine administration on the CNS dopaminergic system in Wistar-Kyoto and spontaneously hypertensive rats: I. Levels of dopamine and metabolites

Effects of acute and subacute cocaine administration on dopamine (DA) and its metabolites in striata and nucleus accumbens of nine week-old Wistar-Kyoto and spontaneously hypertensive rats were studied. Levels of DA,3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined by HPLC-EC. There were no differences in DA levels in striata and nucleus accumbens between control WKY and SHR. Levels of DA in two brain regions were unaffected in groups treated acutely with cocaine. Both strains showed a significant increase in striatal HVA 2 hr after cocaine injection. Seven day treatment declined DA levels in striatum of WKY and in nucleus accumbens of SHR. However, only WKY treated subacutely with cocaine showed significantly increased HVA either with or without changes in DOPAC in nucleus accumbens and striatum, respectively. Increased DOPAC/DA and HVA/DA ratios appeared only in striatum of WKY and in nucleus accumbens of SHR following subacute treatment. These results suggest that subacute cocaine administration affects DA levels in striata and nucleus accumbens differently between WKY and SHR.