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Papers by Frederick Mendelsohn
Corticotropin-releasing factor receptors in rat pituitary gland: autoradiographic localization
Brain Research, 1984
Corticotropin-releasing factor (CRF) receptors were localized in rat pituitary gland by an in vit... more Corticotropin-releasing factor (CRF) receptors were localized in rat pituitary gland by an in vitro labeling light microscopic autoradiographic technique using the radioiodinate ovine CRF analog, [Nle21,125I-Tyr32]-CRF. Our data confirms the existence of CRF receptors in anterior pituitary and demonstrates the presence of specific CRF binding sites in the intermediate lobe of rat pituitary. The distribution of CRF binding sites closely resembles the clustering of corticotrophs in anterior lobe and the uniform distribution of opiomelanocortin-producing cells in the intermediate pituitary. These results suggest that endogenous CRF plays a physiological role in regulating secretion of pro-opiomelanocortin-derived peptides from both anterior and intermediate lobes of rat pituitary.
Kidney International, Dec 1, 1987
Localization and characterization of renal calcitonin receptors by in vitro autoradiography. Calc... more Localization and characterization of renal calcitonin receptors by in vitro autoradiography. Calcitonin receptor binding sites were identified in renal cortex and medulla using the radioligand '251-salmon calcitonin. Microscopic localization of these receptors revealed binding over meduilary and cortical thick ascending limb of the loop of Henle and in distal convoluted tubule. A number of receptor positive cells in the inner medulla were also identified. Characterization of the binding demonstrated a single class of high-affinity binding sites in both the medulla and the cortex with affinity constants of 0.74 0.09 X iOM1 and 0.32 0.05 x l0 M1, respectively, and receptor concentrations of 205 45 fmollmg protein and 453 54 fmol/mg protein, respectively. Competition for '251-salmon calcitonin binding by a wide range of calcitonin analogs revealed a close correspondence between the reported biological potencies and activities in the current system. The localization of binding sites within the nephron corresponds to the reported localization of calcitonin-stimulated adenylate cyclase activity and suggests that the receptor mediated actions of calcitonin in the kidney utilize cyclic AMP as a second messenger. In addition, the microscopic identification of specific calcitonin receptors helps the delineation of direct actions of this hormone from those which are indirect. Calcitonin (CT) is one of the major calcitropic hormones with its primary effect of inhibiting bone resorption by acting on the osteoclast where abundant receptors for calcitonin have been identified [I]. In the kidney, calcitonin affects the handling of Mg2, P042, Ca and Na [2-91, and is also reported to influence the metabolism of Vitamin D in this organ . Receptors for CT have been identified in renal plasma membranes and in cultured kidney cells . In addition, the calcitonin-sensitive adenylate cyclase in the nephron has been well characterized . However, it has been suggested that calcitonin may act independently of adenylate cyclase to influence the metabolism of Vitamin D in the proximal tubule . Recently, CT binding sites in both the cortex and outer medulla have been identified , although without detailed microscopic localization. In vivo autoradiographic studies have localized specific cortical binding sites to distal convoluted
Biochemical Journal, 1991
In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep ... more In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of '25I-labelled salmon CT (1251I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with fl-octyl glucoside. 125I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 +0.5 nm and a maximum binding site concentration of 6.2+ 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems. Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mm. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 1251-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40 % and 46 % dissociation after 2 h at 4 °C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125I-[Arg11'18,4-azidobenzoyl-Lys14]sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of Mr -72000 in both solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified, one of Mr -. 71 000 and another of Mr -88000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues. Abbreviations used: CT, calcitonin (the prefixes s, h and p refer to salmon, human and pig respectively); CGRP, calcitonin-gene-related peptide; PMSF, phenylmethanesulphonyl fluoride; PEG, poly(ethylene glycol).
Hypertension, Jun 1, 1994
sites. Renal vasodilatation induced by sodium nitroprusside or use of the radiolabeled antagonist... more sites. Renal vasodilatation induced by sodium nitroprusside or use of the radiolabeled antagonist analogue l25 I-[ Sar Jle'Jangiotensin II did not alter binding to the inner stripe. In contrast, chronic salt loading or inhibition of angiotensin-converting enzyme by perindopril significantly increased binding not only to the cortical sites but also to the sites in the inner stripe of the outer medulla. Electron microscopic autoradiographs of the inner stripe detected binding in the interstitial cells only in rats treated with chronic salt loading or perindopril. These results suggest that endogenous angiotensins may modulate binding of circulating angiotensin II to the interstitial cells in vivo, and these angiotensin II receptor-bearing cells are more likely to be more responsive to interstitial angiotensin II than to the circulating hormone. (Hypertension. 1994^3 [part 2]:838-843.)
Journal of Clinical Investigation, Aug 1, 1986
Calcitonin receptors have been characterized for the first time in isolated osteoclasts. These re... more Calcitonin receptors have been characterized for the first time in isolated osteoclasts. These receptors have been demonstrated by autoradiographic and biochemical methods, and the cells have also been shown to respond to calcitonin with a dose-dependent increase in cyclic AMP. The receptors in rat osteoclasts are specific and of high affinity (dissociation constant, Kd, 1 to 6 X 10-10 M), and are present in greater numbers than in any cell previously studied (>10' per cell). Chemical cross-linking of "SIlabeled salmon calcitonin to osteoclasts using disuccinimidyl suberate resulted in identification of a receptor component with a relative molecular weight of 80,000-90,000. By counting grains in autoradiographic experiments, we found that >80% of spe- cifically bound radioactivity was associated with multinucleate osteoclasts and the remainder was associated with mononuclear cells that are not osteoblasts, but that may be osteoclast pre- cursors.
Journal of Neurochemistry, Jan 18, 2002
Local Actions of Angiotensin II
Journal of Cardiovascular Pharmacology, 1986
ABSTRACT
Measurement of angiotensin converting enzyme induction and inhibition using quantitative in vitro autoradiography: tissue selective induction after chronic lisinopril treatment
Journal of Hypertension, Jul 1, 1991
Angiotensin converting enzyme (ACE) inhibitors lead to induction of ACE in animals and humans. Th... more Angiotensin converting enzyme (ACE) inhibitors lead to induction of ACE in animals and humans. This complicates the use of ACE enzymatic activity as an index of inhibition in plasma or tissues after chronic administration of ACE inhibitors. We have, therefore, developed a method for ACE measurement by in vitro autoradiography using an 125I-labelled inhibitor to quantitate total ACE and the concentration of free (not inhibited) ACE in tissues after prolonged administration of ACE inhibitors to rats. Measurements made on unprocessed tissue sections reflect residual free ACE activity in the presence of the unlabelled inhibitor. In a parallel series of adjacent sections, the ACE inhibitor is dissociated from the enzyme by reversibly denaturing the enzyme by zinc chelation. This is followed by reconstitution of the active enzyme by zinc ion replacement and measuring total enzyme concentration. This technique permits measurement of the extent of ACE inhibition and induction. This method was evaluated in tissues of rats following chronic oral administration of lisinopril (10 mg/kg per day) for 2 weeks. The pattern of ACE inhibition was similar to that seen in our previous acute studies. However, induction of ACE was found to be organ specific; plasma total ACE increased 1.75-fold and total ACE in the lung increased by 30% compared with untreated animals, but there was no demonstrable change in total ACE concentration in the kidney, adrenal or aorta. Despite this, during chronic treatment with lisinopril, ACE activity in all of these organs was inhibited with low levels of free ACE.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypertension, Mar 1, 1988
Inhibition of angiotensin converting enzyme (ACE) in serum and tissues of rats was studied after ... more Inhibition of angiotensin converting enzyme (ACE) in serum and tissues of rats was studied after administration of lisinopril, an ACE inhibitor. Tissue ACE was assessed by quantitative in vitro autoradiography using the ACE inhibitor [ I25 I]351A, as a ligand, and serum ACE was measured by a fluorimetric method. Following oral administration of lisinopril (10 mg/kg), serum ACE activity was acutely reduced but recovered gradually over 24 hours. Four hours after lisinopril administration, ACE activity was markedly inhibited in kidney (11% of control level), adrenal (8%), duodenum (8%), and lung (33%; p<0.05). In contrast, ACE in testis was little altered by lisinopril (96%). In brain, ACE activity was markedly reduced 4 hours after lisinopril administration in the circumventricular organs, including the subfornical organ (16-22%) and organum vasculosum of the lamina terminalis (7%; p < 0.05). In other areas of the brain, including the choroid plexus and caudate putamen, ACE activity was unchanged. Twenty-four hours after administration, ACE activity in peripheral tissues and the circumventricular organs of the brain had only partially recovered toward control levels, as it was still below 50% of control activity levels. These results establish that lisinopril has differential effects on inhibiting ACE in different tissues and suggest that the prolonged tissue ACE inhibition after a single oral dose of lisinopril may reflect targets involved in the hypotensive action of ACE inhibitors. (Hypertension 11: 230-238, 1988) KEY WORDS • angiotensin converting enzyme inhibitors • hypotensive action lisinopril • blood-brain barrier • renin ANGIOTENSIN converting enzyme (ACE; / \ EC3.4.15.1) is a peptidyl dipeptidase that is X \. responsible for the conversion of angiotensin I to angiotensin II. High concentrations of ACE are found in vascular endothelial cells, 1 renal proximal tubules, 2 intestinal mucosa, 3 the male reproductive system, 4 and some specific areas of the brain. 5-6 Recently, ACE inhibitors such as captopril and enalapril have been found to be effective for the treatment of hypertension and congestive heart failure. 7 The mechanism of the hypotensive action of these drugs is unclear. Although the circulating renin-angiotensin system was initially considered the primary target for ACE inhibitors, this possibility must be reevaluated
European Journal of Pharmacology, 1989
Endothelin (ET) is one of the most potent known vasoconstrictors (Yanagisawa et al., 1988). Howev... more Endothelin (ET) is one of the most potent known vasoconstrictors (Yanagisawa et al., 1988). However its physiological role and distribution of its receptors are still unknown. In the present study the radioligand, 125I-endothelin, was used to map receptors for ET in rat kidney using in vitro autoradiography and computerized densitometry (Mendelsohn et al., 1987). Porcine endothelin (Protein Research Foundation, Osaka, Japan) was radioiodinated with ~25I using Iodogen and purified by gradient elution from C18 cartridges. Autoradiographs were generated by incubating 20 /tm frozen sections of rat kidneys with 0.3 /~Ci/ml (= 20 pM) 125I-ET in 20 mM HEPES buffer, pH 7.4, containing 135 mM NaC1, 2 mM CaC12, 0.2% bovine serum albumin and 0.01% bacitracin for 1 h at 20 o C. Nonspecific binding was determined in the presence of 1 /.tM unlabelled ET. After incubation, the sections were washed, and exposed to X-ray film for 12-24 h at room temperature. Optical density of the X-ray films was quantitated by computerized densitometry (Mendelsohn et al., 1987). A very high density of ET-receptor binding occurs overlying glomeruli in the cortex, and over the inner medulla (fig. 1). A high density of binding occurs over longitudinal bands in the inner stripe of outer medulla, corresponding to vasa recta bundles, and also in the inter bundle zone of
Localization and Quantitation of Active Renin in Monkey Kidney by Radioinhibitor Binding and In Vitro Autoradiography
American Journal of Hypertension, Jun 1, 1994
ABSTRACT We developed an in vitro autoradiographic method to localize and quantify active renin i... more ABSTRACT We developed an in vitro autoradiographic method to localize and quantify active renin in primate tissues. Active renin in monkey kidney sections was labeled with the primate specific renin inhibitor, 3H-CGP29287, and quantitated with autoradiography and computerized densitometry. Microscopic emulsion autoradiography was carried out to clarify the detailed localization of the binding. Non-specific binding to aspartyl proteases other than renin was blocked using 1 mumol/L of N-acetyl-pepstatin. To assess the usefulness of this procedure, binding of 3H-CGP29287 was examined both by film and emulsion autoradiography in the kidneys of monkeys (Macaca fuscata) that were given chronically either an angiotensin converting enzyme inhibitor (trandolapril), an angiotensin II receptor antagonist (E4177), or vehicle. 3H-CGP29287 was found to bind very selectively to the juxtaglomerular apparatus (JGA) under control conditions. In monkeys treated with trandolapril or E4177, 3H-CGP29287 binding was increased in proportion to the increase in renal renin concentration determined enzymatically; in these kidneys, emulsion autoradiography revealed radioinhibitor binding extending far from the JGA. The potency of a series of unlabeled renin inhibitor in competing for 3H-CGP29287 binding in the autoradiographic system closely paralleled their potencies, as determined in inhibiting renin by an enzymatic assay. This technique permits specific labeling of the catalytic site of renin in the monkey kidney sections.
Actions of Angiotensin II in the Ventrolateral Medulla Oblongata
Birkhäuser Boston eBooks, 1991
Angiotensin II (ANG II) regulates numerous brain functions including drinking and salt appetite, ... more Angiotensin II (ANG II) regulates numerous brain functions including drinking and salt appetite, release of both anterior and posterior pituitary hormones, and regulation of cardiovascular function through interactions with central control of efferent autonomic activity (see Phillips, 1987) as well as the well characterized actions on peripheral tissues (see Peach, 1977). The central actions are probably mediated by both systemic and endogenous neuronal angiotensin.
Action of Angiotensins I, II, and III on Aldosterone Production by Isolated Rat Adrenal Zona Glomerulosa Cells: Importance of Metabolism and Conversion of Peptidesin Vitro*
Endocrinology, Jun 1, 1980
ABSTRACT
Evaluation of Renin and Angiotensin Assays and Their Clinical Application
The Medical Journal of Australia, 1971
A brief review of the biochemistry and physiological functions of the renin‐angiotensin system is... more A brief review of the biochemistry and physiological functions of the renin‐angiotensin system is presented. A critical analysis is given of the various methods, both bioassay and radioimmunoassay, for measuring plasma renin and angiotensin levels. With the development of radioimmunoassay, specific, precise and rapid measurements of renin and angiotensin levels may now be made. Various factors that may influence the plasma level of renin are discussed, and the clinical applications for plasma renin measurements are listed.
Localization of Angiotensin II Receptors in Rat Kidney and Brain
European journal of endocrinology, Jun 1, 1981
Normal human serum markedly stimulated aldosterone production from rat adrenal glomerulosa cells ... more Normal human serum markedly stimulated aldosterone production from rat adrenal glomerulosa cells incubated in Krebs Ringer bicarbonate medium (KRBGA). The effect was dose-related. In [K+] 3.6 mM KRBGA medium, serum stimulated aldosterone output to higher levels than those produced by maximal doses of serotonin (5 HT), angiotensin II (All) or high [K+] (8.4 mM). Cells maximally stimulated by high [K+], 5 HT or All in KRBGA medium were further stimulated by serum. The angiotensin analogue, [Sar1, Ala8]-AII abolished the effect of All but not that of high [K+] or serum. Basal and ACTH-stimulated corticosterone outputs of rat fasciculata cells were not significantly affected by sera known to stimulate glomerulosa cells. Aldosterone stimulating activity of serum was dialysable and fully recovered in a serum ultrafiltrate. The serotonin blockers methysergide and metergoline abolished the aldosterone stimulating activity of serum but also depressed basal aldosterone output and methysergide reduced K+-stimulated output. Chymotrypsin digestion abolished the aldosterone stimulating activity of All but not that of serotonin or serum.
Neuroscience Letters, Jun 1, 1996
There is increasing evidence that neuropeptides have trophic functions during embryogenesis. We e... more There is increasing evidence that neuropeptides have trophic functions during embryogenesis. We examined the ability of angiotensin II, substance P, soraatostatin-28 and luteinising hormone-releasing hormone to influence neurite outgrowth from embryonic chick sympathetic neurons in culture. Nanomolar concentrations of angiotensin II inhibited neurite outgrowth, whereas the other peptides had no effect at similar concentrations. The effect of angiotensin II on neurite outgrowth is likely to be mediated by an atypical angiotensin receptor, as it was only weakly inhibited by [sarl,ala8]angiotensin II, and was not inhibited by losartan, an inhibitor of mammalian AT 1 receptors, or PD123319, an AT 2 inhibitor. Neurite outgrowth was also inhibited by angiotensin III and angiotensin IV but not by angiotensinogen I1_14. The study provides further evidence that angiotensin peptides, like classical neurotransmitters, may have trophic functions during embryogenesis.
Effect of acute and chronic administration of ceronapril on angiotensin converting enzyme in plasma, kidney, lung, brain regions and cerebrospinal fluid of rats
Neuropharmacology, Sep 1, 1992
ABSTRACT
Lack of Response of Sympathetic Nervous System to Angiotensin Infusion
The Lancet, Mar 1, 1980
Corticotropin-releasing factor receptors in rat pituitary gland: autoradiographic localization
Brain Research, 1984
Corticotropin-releasing factor (CRF) receptors were localized in rat pituitary gland by an in vit... more Corticotropin-releasing factor (CRF) receptors were localized in rat pituitary gland by an in vitro labeling light microscopic autoradiographic technique using the radioiodinate ovine CRF analog, [Nle21,125I-Tyr32]-CRF. Our data confirms the existence of CRF receptors in anterior pituitary and demonstrates the presence of specific CRF binding sites in the intermediate lobe of rat pituitary. The distribution of CRF binding sites closely resembles the clustering of corticotrophs in anterior lobe and the uniform distribution of opiomelanocortin-producing cells in the intermediate pituitary. These results suggest that endogenous CRF plays a physiological role in regulating secretion of pro-opiomelanocortin-derived peptides from both anterior and intermediate lobes of rat pituitary.
Kidney International, Dec 1, 1987
Localization and characterization of renal calcitonin receptors by in vitro autoradiography. Calc... more Localization and characterization of renal calcitonin receptors by in vitro autoradiography. Calcitonin receptor binding sites were identified in renal cortex and medulla using the radioligand '251-salmon calcitonin. Microscopic localization of these receptors revealed binding over meduilary and cortical thick ascending limb of the loop of Henle and in distal convoluted tubule. A number of receptor positive cells in the inner medulla were also identified. Characterization of the binding demonstrated a single class of high-affinity binding sites in both the medulla and the cortex with affinity constants of 0.74 0.09 X iOM1 and 0.32 0.05 x l0 M1, respectively, and receptor concentrations of 205 45 fmollmg protein and 453 54 fmol/mg protein, respectively. Competition for '251-salmon calcitonin binding by a wide range of calcitonin analogs revealed a close correspondence between the reported biological potencies and activities in the current system. The localization of binding sites within the nephron corresponds to the reported localization of calcitonin-stimulated adenylate cyclase activity and suggests that the receptor mediated actions of calcitonin in the kidney utilize cyclic AMP as a second messenger. In addition, the microscopic identification of specific calcitonin receptors helps the delineation of direct actions of this hormone from those which are indirect. Calcitonin (CT) is one of the major calcitropic hormones with its primary effect of inhibiting bone resorption by acting on the osteoclast where abundant receptors for calcitonin have been identified [I]. In the kidney, calcitonin affects the handling of Mg2, P042, Ca and Na [2-91, and is also reported to influence the metabolism of Vitamin D in this organ . Receptors for CT have been identified in renal plasma membranes and in cultured kidney cells . In addition, the calcitonin-sensitive adenylate cyclase in the nephron has been well characterized . However, it has been suggested that calcitonin may act independently of adenylate cyclase to influence the metabolism of Vitamin D in the proximal tubule . Recently, CT binding sites in both the cortex and outer medulla have been identified , although without detailed microscopic localization. In vivo autoradiographic studies have localized specific cortical binding sites to distal convoluted
Biochemical Journal, 1991
In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep ... more In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of '25I-labelled salmon CT (1251I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with fl-octyl glucoside. 125I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 +0.5 nm and a maximum binding site concentration of 6.2+ 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems. Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mm. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 1251-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40 % and 46 % dissociation after 2 h at 4 °C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125I-[Arg11'18,4-azidobenzoyl-Lys14]sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of Mr -72000 in both solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified, one of Mr -. 71 000 and another of Mr -88000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues. Abbreviations used: CT, calcitonin (the prefixes s, h and p refer to salmon, human and pig respectively); CGRP, calcitonin-gene-related peptide; PMSF, phenylmethanesulphonyl fluoride; PEG, poly(ethylene glycol).
Hypertension, Jun 1, 1994
sites. Renal vasodilatation induced by sodium nitroprusside or use of the radiolabeled antagonist... more sites. Renal vasodilatation induced by sodium nitroprusside or use of the radiolabeled antagonist analogue l25 I-[ Sar Jle'Jangiotensin II did not alter binding to the inner stripe. In contrast, chronic salt loading or inhibition of angiotensin-converting enzyme by perindopril significantly increased binding not only to the cortical sites but also to the sites in the inner stripe of the outer medulla. Electron microscopic autoradiographs of the inner stripe detected binding in the interstitial cells only in rats treated with chronic salt loading or perindopril. These results suggest that endogenous angiotensins may modulate binding of circulating angiotensin II to the interstitial cells in vivo, and these angiotensin II receptor-bearing cells are more likely to be more responsive to interstitial angiotensin II than to the circulating hormone. (Hypertension. 1994^3 [part 2]:838-843.)
Journal of Clinical Investigation, Aug 1, 1986
Calcitonin receptors have been characterized for the first time in isolated osteoclasts. These re... more Calcitonin receptors have been characterized for the first time in isolated osteoclasts. These receptors have been demonstrated by autoradiographic and biochemical methods, and the cells have also been shown to respond to calcitonin with a dose-dependent increase in cyclic AMP. The receptors in rat osteoclasts are specific and of high affinity (dissociation constant, Kd, 1 to 6 X 10-10 M), and are present in greater numbers than in any cell previously studied (>10' per cell). Chemical cross-linking of "SIlabeled salmon calcitonin to osteoclasts using disuccinimidyl suberate resulted in identification of a receptor component with a relative molecular weight of 80,000-90,000. By counting grains in autoradiographic experiments, we found that >80% of spe- cifically bound radioactivity was associated with multinucleate osteoclasts and the remainder was associated with mononuclear cells that are not osteoblasts, but that may be osteoclast pre- cursors.
Journal of Neurochemistry, Jan 18, 2002
Local Actions of Angiotensin II
Journal of Cardiovascular Pharmacology, 1986
ABSTRACT
Measurement of angiotensin converting enzyme induction and inhibition using quantitative in vitro autoradiography: tissue selective induction after chronic lisinopril treatment
Journal of Hypertension, Jul 1, 1991
Angiotensin converting enzyme (ACE) inhibitors lead to induction of ACE in animals and humans. Th... more Angiotensin converting enzyme (ACE) inhibitors lead to induction of ACE in animals and humans. This complicates the use of ACE enzymatic activity as an index of inhibition in plasma or tissues after chronic administration of ACE inhibitors. We have, therefore, developed a method for ACE measurement by in vitro autoradiography using an 125I-labelled inhibitor to quantitate total ACE and the concentration of free (not inhibited) ACE in tissues after prolonged administration of ACE inhibitors to rats. Measurements made on unprocessed tissue sections reflect residual free ACE activity in the presence of the unlabelled inhibitor. In a parallel series of adjacent sections, the ACE inhibitor is dissociated from the enzyme by reversibly denaturing the enzyme by zinc chelation. This is followed by reconstitution of the active enzyme by zinc ion replacement and measuring total enzyme concentration. This technique permits measurement of the extent of ACE inhibition and induction. This method was evaluated in tissues of rats following chronic oral administration of lisinopril (10 mg/kg per day) for 2 weeks. The pattern of ACE inhibition was similar to that seen in our previous acute studies. However, induction of ACE was found to be organ specific; plasma total ACE increased 1.75-fold and total ACE in the lung increased by 30% compared with untreated animals, but there was no demonstrable change in total ACE concentration in the kidney, adrenal or aorta. Despite this, during chronic treatment with lisinopril, ACE activity in all of these organs was inhibited with low levels of free ACE.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypertension, Mar 1, 1988
Inhibition of angiotensin converting enzyme (ACE) in serum and tissues of rats was studied after ... more Inhibition of angiotensin converting enzyme (ACE) in serum and tissues of rats was studied after administration of lisinopril, an ACE inhibitor. Tissue ACE was assessed by quantitative in vitro autoradiography using the ACE inhibitor [ I25 I]351A, as a ligand, and serum ACE was measured by a fluorimetric method. Following oral administration of lisinopril (10 mg/kg), serum ACE activity was acutely reduced but recovered gradually over 24 hours. Four hours after lisinopril administration, ACE activity was markedly inhibited in kidney (11% of control level), adrenal (8%), duodenum (8%), and lung (33%; p<0.05). In contrast, ACE in testis was little altered by lisinopril (96%). In brain, ACE activity was markedly reduced 4 hours after lisinopril administration in the circumventricular organs, including the subfornical organ (16-22%) and organum vasculosum of the lamina terminalis (7%; p < 0.05). In other areas of the brain, including the choroid plexus and caudate putamen, ACE activity was unchanged. Twenty-four hours after administration, ACE activity in peripheral tissues and the circumventricular organs of the brain had only partially recovered toward control levels, as it was still below 50% of control activity levels. These results establish that lisinopril has differential effects on inhibiting ACE in different tissues and suggest that the prolonged tissue ACE inhibition after a single oral dose of lisinopril may reflect targets involved in the hypotensive action of ACE inhibitors. (Hypertension 11: 230-238, 1988) KEY WORDS • angiotensin converting enzyme inhibitors • hypotensive action lisinopril • blood-brain barrier • renin ANGIOTENSIN converting enzyme (ACE; / \ EC3.4.15.1) is a peptidyl dipeptidase that is X \. responsible for the conversion of angiotensin I to angiotensin II. High concentrations of ACE are found in vascular endothelial cells, 1 renal proximal tubules, 2 intestinal mucosa, 3 the male reproductive system, 4 and some specific areas of the brain. 5-6 Recently, ACE inhibitors such as captopril and enalapril have been found to be effective for the treatment of hypertension and congestive heart failure. 7 The mechanism of the hypotensive action of these drugs is unclear. Although the circulating renin-angiotensin system was initially considered the primary target for ACE inhibitors, this possibility must be reevaluated
European Journal of Pharmacology, 1989
Endothelin (ET) is one of the most potent known vasoconstrictors (Yanagisawa et al., 1988). Howev... more Endothelin (ET) is one of the most potent known vasoconstrictors (Yanagisawa et al., 1988). However its physiological role and distribution of its receptors are still unknown. In the present study the radioligand, 125I-endothelin, was used to map receptors for ET in rat kidney using in vitro autoradiography and computerized densitometry (Mendelsohn et al., 1987). Porcine endothelin (Protein Research Foundation, Osaka, Japan) was radioiodinated with ~25I using Iodogen and purified by gradient elution from C18 cartridges. Autoradiographs were generated by incubating 20 /tm frozen sections of rat kidneys with 0.3 /~Ci/ml (= 20 pM) 125I-ET in 20 mM HEPES buffer, pH 7.4, containing 135 mM NaC1, 2 mM CaC12, 0.2% bovine serum albumin and 0.01% bacitracin for 1 h at 20 o C. Nonspecific binding was determined in the presence of 1 /.tM unlabelled ET. After incubation, the sections were washed, and exposed to X-ray film for 12-24 h at room temperature. Optical density of the X-ray films was quantitated by computerized densitometry (Mendelsohn et al., 1987). A very high density of ET-receptor binding occurs overlying glomeruli in the cortex, and over the inner medulla (fig. 1). A high density of binding occurs over longitudinal bands in the inner stripe of outer medulla, corresponding to vasa recta bundles, and also in the inter bundle zone of
Localization and Quantitation of Active Renin in Monkey Kidney by Radioinhibitor Binding and In Vitro Autoradiography
American Journal of Hypertension, Jun 1, 1994
ABSTRACT We developed an in vitro autoradiographic method to localize and quantify active renin i... more ABSTRACT We developed an in vitro autoradiographic method to localize and quantify active renin in primate tissues. Active renin in monkey kidney sections was labeled with the primate specific renin inhibitor, 3H-CGP29287, and quantitated with autoradiography and computerized densitometry. Microscopic emulsion autoradiography was carried out to clarify the detailed localization of the binding. Non-specific binding to aspartyl proteases other than renin was blocked using 1 mumol/L of N-acetyl-pepstatin. To assess the usefulness of this procedure, binding of 3H-CGP29287 was examined both by film and emulsion autoradiography in the kidneys of monkeys (Macaca fuscata) that were given chronically either an angiotensin converting enzyme inhibitor (trandolapril), an angiotensin II receptor antagonist (E4177), or vehicle. 3H-CGP29287 was found to bind very selectively to the juxtaglomerular apparatus (JGA) under control conditions. In monkeys treated with trandolapril or E4177, 3H-CGP29287 binding was increased in proportion to the increase in renal renin concentration determined enzymatically; in these kidneys, emulsion autoradiography revealed radioinhibitor binding extending far from the JGA. The potency of a series of unlabeled renin inhibitor in competing for 3H-CGP29287 binding in the autoradiographic system closely paralleled their potencies, as determined in inhibiting renin by an enzymatic assay. This technique permits specific labeling of the catalytic site of renin in the monkey kidney sections.
Actions of Angiotensin II in the Ventrolateral Medulla Oblongata
Birkhäuser Boston eBooks, 1991
Angiotensin II (ANG II) regulates numerous brain functions including drinking and salt appetite, ... more Angiotensin II (ANG II) regulates numerous brain functions including drinking and salt appetite, release of both anterior and posterior pituitary hormones, and regulation of cardiovascular function through interactions with central control of efferent autonomic activity (see Phillips, 1987) as well as the well characterized actions on peripheral tissues (see Peach, 1977). The central actions are probably mediated by both systemic and endogenous neuronal angiotensin.
Action of Angiotensins I, II, and III on Aldosterone Production by Isolated Rat Adrenal Zona Glomerulosa Cells: Importance of Metabolism and Conversion of Peptidesin Vitro*
Endocrinology, Jun 1, 1980
ABSTRACT
Evaluation of Renin and Angiotensin Assays and Their Clinical Application
The Medical Journal of Australia, 1971
A brief review of the biochemistry and physiological functions of the renin‐angiotensin system is... more A brief review of the biochemistry and physiological functions of the renin‐angiotensin system is presented. A critical analysis is given of the various methods, both bioassay and radioimmunoassay, for measuring plasma renin and angiotensin levels. With the development of radioimmunoassay, specific, precise and rapid measurements of renin and angiotensin levels may now be made. Various factors that may influence the plasma level of renin are discussed, and the clinical applications for plasma renin measurements are listed.
Localization of Angiotensin II Receptors in Rat Kidney and Brain
European journal of endocrinology, Jun 1, 1981
Normal human serum markedly stimulated aldosterone production from rat adrenal glomerulosa cells ... more Normal human serum markedly stimulated aldosterone production from rat adrenal glomerulosa cells incubated in Krebs Ringer bicarbonate medium (KRBGA). The effect was dose-related. In [K+] 3.6 mM KRBGA medium, serum stimulated aldosterone output to higher levels than those produced by maximal doses of serotonin (5 HT), angiotensin II (All) or high [K+] (8.4 mM). Cells maximally stimulated by high [K+], 5 HT or All in KRBGA medium were further stimulated by serum. The angiotensin analogue, [Sar1, Ala8]-AII abolished the effect of All but not that of high [K+] or serum. Basal and ACTH-stimulated corticosterone outputs of rat fasciculata cells were not significantly affected by sera known to stimulate glomerulosa cells. Aldosterone stimulating activity of serum was dialysable and fully recovered in a serum ultrafiltrate. The serotonin blockers methysergide and metergoline abolished the aldosterone stimulating activity of serum but also depressed basal aldosterone output and methysergide reduced K+-stimulated output. Chymotrypsin digestion abolished the aldosterone stimulating activity of All but not that of serotonin or serum.
Neuroscience Letters, Jun 1, 1996
There is increasing evidence that neuropeptides have trophic functions during embryogenesis. We e... more There is increasing evidence that neuropeptides have trophic functions during embryogenesis. We examined the ability of angiotensin II, substance P, soraatostatin-28 and luteinising hormone-releasing hormone to influence neurite outgrowth from embryonic chick sympathetic neurons in culture. Nanomolar concentrations of angiotensin II inhibited neurite outgrowth, whereas the other peptides had no effect at similar concentrations. The effect of angiotensin II on neurite outgrowth is likely to be mediated by an atypical angiotensin receptor, as it was only weakly inhibited by [sarl,ala8]angiotensin II, and was not inhibited by losartan, an inhibitor of mammalian AT 1 receptors, or PD123319, an AT 2 inhibitor. Neurite outgrowth was also inhibited by angiotensin III and angiotensin IV but not by angiotensinogen I1_14. The study provides further evidence that angiotensin peptides, like classical neurotransmitters, may have trophic functions during embryogenesis.
Effect of acute and chronic administration of ceronapril on angiotensin converting enzyme in plasma, kidney, lung, brain regions and cerebrospinal fluid of rats
Neuropharmacology, Sep 1, 1992
ABSTRACT
Lack of Response of Sympathetic Nervous System to Angiotensin Infusion
The Lancet, Mar 1, 1980