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J W Harding

Publications and source records attributed to J W Harding.

At least 91 records · Page 5Linked to original sources
PubMed1988

Differential effects of aminopeptidase inhibitors on angiotensin-induced pressor responses.

Recent iontophoretic data suggest that conversion of angiotensin II (AII) to angiotensin III (AIII) may be necessary before the peptide can activate central angiotensin-sensitive neurons. Furthermore, this conversion may be inhibited by the aminopeptidase A inhibitor, amastatin. In the present study we investigated the importance of aminopeptidase activity on central angiotensin-induced pressor responses. Intracerebroventricular (i.c.v.) pretreatment with amastatin, suppressed i.c.v. AII-induced pressor responses. Pretreatment with the aminopeptidase B inhibitor, bestatin, increased pressor responses to AIII. Pressor responses induced by the aminopeptidase-resistant analogue, [Sar1]angiotensin II, were not affected by pretreatment with angiotensin inhibitors. These results support the hypothesis that AII must be converted to AIII to be active in the brain.

Aminopeptidases↗
PubMed1988

Tachyphylaxis of dipsogenic activity to intracerebroventricular administration of angiotensins.

Repeated intracerebroventricular (i.c.v.) administration of angiotensin II (AII) and angiotensin III (AIII) induced dipsogenic tachyphylaxis in the Sprague-Dawley rat. AIII caused a rapid suppression of drinking, whereas AII showed a progressive decline of water consumption with repeated injections. Tachyphylaxis due to the repeated i.c.v. application of AII failed to abolish subsequent drinking induced by neurotensin or carbachol, suggesting that the tachyphylaxis may be specific to the angiotensinergic system. However, AII-induced tachyphylaxis caused a complete elimination of bestatin-induced drinking which was anticipated given the likelihood that this aminopeptidase B inhibitor has its dipsogenic effect by inhibiting degradation of endogenous angiotensins. Angiotensin-induced tachyphylaxis responses could not be attributed to diminished dipsogenic activity due to volemic expansion of either the cerebroventricular space or gastrointestinal tract. These results concerned with central tachyphylaxis are consistent with previous findings in the periphery and suggest that desensitization of angiotensin receptors occurs in both populations.

Angiotensin II↗
PubMed1988

Central effects of a specific angiotensin receptor antagonist, sarthran (Sar1, Thr8AII) in normotensive and spontaneously hypertensive rat strains.

The specific angiotensin receptor antagonist, Sar1, Thr8AII (sarthran), was infused intracerebroventricularly in alert spontaneously hypertensive rats (SHR), and Wistar-Kyoto (WKY) and Sprague-Dawley (SD) normotensive rat strains. This resulted in a mean decrease of 35 mm Hg in the SHR group by 25 min post-infusion, and corresponding decreases in the WKY and SD rats of 13 and 15 mm Hg, respectively. A prominent transient sarthran-induced elevation in blood pressure was noted in the SHR group during the 5-min infusion. This agonistic effect was not observed in members of the WKY and SD strains. These data encourage the use of sarthran as a valuable pharmacological probe in the examination of the role of the brain renin-angiotensin system in hypertension.

Angiotensin II↗
PubMed1988

Effects of aminopeptidase inhibition on the half-lives of [125I]angiotensins in the cerebroventricles of the rat.

Anesthetized Sprague-Dawley rats fitted with intracerebroventricular (i.c.v.) cannulas were infused with one of the aminopeptidase inhibitors, amastatin or bestatin, over a 5-min period. After infusion, 1-2 X 10(6) cpm of [125I]angiotensin II ([125I]AII) or [125I]angiotensin III ([125I]AIII) was injected through the same cannula. The rats were subsequently killed 60 s later by focused microwave irradiation which instantaneously terminated further [125I]angiotensin metabolism. HPLC analysis of the extracted [125I]angiotensin and metabolic products allowed for the calculation of t1/2s of disappearance for the parent peptides. Both inhibitors effectively lengthened the half-lives of [125I]AII and [125I]AIII. Bestatin, which is considered a selective aminopeptidase B blocker, had a more pronounced effect on [125I]AIII metabolism, while amastatin, a selective aminopeptidase A inhibitor, was better at slowing [125I]AII degradation. The results indicate that amastatin and bestatin are very effective blockers of the cerebroventricular metabolism of angiotensins but are only marginally selective with regard to AII and AIII.

Aminopeptidases↗
PubMed1988

Elevated salt appetite and brain binding of angiotensin II in mineralocorticoid-treated rats.

Angiotensin II (Ang II) and aldosterone levels increase with sodium deficiency, promoting sodium conservation and arousing a salt appetite in rats. The mechanism(s), by which these two hormones interact to produce salt appetite is not known. The experiments reported here tested the possibility that increased mineralocorticoids change the number and/or affinity of Ang receptors in the brain. Rats were given a series of deoxycorticosterone acetate (DOCA) injections (500 micrograms/day, s.c., for 4 days) which are known to produce a salt appetite when given in conjunction with an intracerebroventricular injection of Ang. The binding of 125I-Ang II to membranes prepared from the septal-anteroventral third ventricular region was then examined. DOCA treatment resulted in a significant increase in the number of Ang binding sites (Bmax) with no change in binding affinity (Kd). The binding of 125I-Ang II was then investigated in membranes prepared from 12 other brain regions as well as the pituitary and adrenal gland, showing that the increase in binding capacity occurred in only a few specific brain regions. A third experiment verified that the DOCA treatment used here was sufficient to arouse a salt appetite when combined with a single intracerebroventricular injection of Ang II. The mechanism that underlies the production of salt appetite by aldosterone and Ang II may at least partially consist of mineralocorticoid-induced increases in the number of Ang receptors in discrete brain regions.

Angiotensin II↗
PubMed1988

Influence of aminopeptidase inhibitors on brain angiotensin metabolism and drinking in rats.

The metabolism of [125I]angiotensin II (AII, octopeptide) and [125I]angiotensin III (AIII, heptapeptide) was determined in the cerebroventricular compartment following intracerebroventricular (i.c.v.) pretreatment with artificial cerebrospinal fluid (aCSF) or the aminopeptidase B inhibitor, bestatin (BE). Microwave fixation was used to stop brain aminopeptidase activity followed by high-performance liquid chromatography methods to measure degradation. The resulting mean t 1/2 values were: aCSF/AII = 23.5 s, BE/AII = 32.0 s; aCSF/AIII = 8.2 s, BE/AIII = 16.0 s. A second experiment indicated that i.c.v. administered BE induced considerable water consumption, while amastatin (AM, an aminopeptidase A inhibitor) did not. Combined i.c.v. treatment with BE/AII or BE/AIII produced water consumptions that were equivalent with the sum of the water intakes due to independent treatments. Unexpectedly, combined treatment with AM/AII also significantly facilitated water consumption as compared with the independent treatments. These results suggest that i.c.v. BE-induced drinking is due to the extension of the half-life of endogenous AIII, and that both AM and BE facilitate drinking to i.c.v. AII and AIII by inhibiting the degradation of AII and AIII. This delay in degradation prolongs the action of AIII at central angiotensin receptors while also slowing the conversion of AII to AIII, thus providing a depot of available AII to be converted to AIII in an obligatory manner.

Aminopeptidases↗
PubMed1988

Heightened blood pressure responsiveness to intracarotid infusion of angiotensins in the spontaneously hypertensive rat.

The purpose of this study was to test the hypothesis that intracarotid infusion of angiotensin via a brachial arterial catheter results in a heightened pressor response in the alert spontaneously hypertensive rat (SHR) as previously observed for intracerebroventricular (ICV) injection of angiotensin. We infused angiotensin II and III since these ligands are equivalently potent with respect to peak pressor effect when delivered ICV. We measured somewhat greater pressor responsiveness to AII than to AIII in the Wistar-Kyoto (WKY) normotensive control strain from a baselevel of 133.1 +/- 5.8 (mean +/- SEM) to 151.3 +/- 6.2 mmHg (+13.7%) at the 100 pmol/kg/min dose of AII, and from 132.5 +/- 5.8 to 146.0 +/- 6.1 mmHg (+10.2%) for AIII. The SHR revealed a heightened pressor sensitivity to AII, from a baselevel of 170.0 +/- 3.8 to 200.6 +/- 5.9 mmHg (+18%) while the response to AIII was less dramatic, from 171.3 +/- 2.1 to 189.8 +/- 2.4 mmHg (+10.8%). These findings suggest that a similar heightened pressor responsiveness occurs to peripheral infusion of angiotensin II in the SHR as previously observed to ICV injection.

Angiotensin II↗
PubMed1988

Pressor responses to amastatin, bestatin and Plummer's inhibitors are suppressed by pretreatment with the angiotensin receptor antagonist sarthran.

The aminopeptidase inhibitors, amastatin (AM) and bestatin (BE), and carboxypeptidase inhibitor Plummer's (PL) were applied intracerebroventricularly (ICV) in rats following pretreatment with the angiotensin receptor antagonist sarthran (Sar1,Thr8-AII) or artificial cerebrospinal fluid. Angiotensin II (AII) was also included as a comparison vasoactive peptide. Pressor responses were recorded at 30 min intervals for 90 min to ascertain the duration of the antagonistic effect of sarthran on subsequent injections of AM, BE, PL and AII. Sarthran was effective in suppressing pressor activity to AII- and PL-induced pressor activity until 60 min following pretreatment, and AM- and BE-induced pressor responses until 90 min following pretreatment. These data suggest that AM, BE and PL are having their pressor effects via the central angiotensinergic system and that the patterns of AM, BE, PL and AII recovery from the influence of a specific angiotensin receptor antagonist are similar. The results are consistent with the concept that these inhibitors may increase endogenously synthesized angiotensins which are associated with pressor responses.

Aminopeptidases↗
PubMed1988

Inability of [125I]Sar1, Ile8-angiotensin II to move between the blood and cerebrospinal fluid compartments.

The angiotensin II competitive antagonist [125I]-Sar1, Ile8-angiotensin II was not transported from the vascular space to the cerebroventricular space in either intact or nephrectomized rats. In addition [125I]Sar1, Ile8-angiotensin II lacked the capacity to move in the opposite direction over a 20-min collection period following cerebroventricular infusion. These data suggest that angiotensins lack the capacity to move freely between the blood and cerebrospinal fluid compartments and are consistent with the notion that blood-borne and cerebroventricular angiotensins access different receptor populations.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1988

Comparison of 125I-angiotensin III and 125I-angiotensin II binding to rat brain membranes.

The binding of 125I-angiotensin III (125I-ANG III) to rat brain membranes was examined and compared with that of 125I-angiotensin II (125I-ANG II). Degradation of each ligand, as monitored by HPLC, was effectively inhibited using fragments of ANG III and ANG II known to have little affinity for angiotensin binding sites. Three classes of 125I-ANG III-binding sites were observed based on affinity (KD = 0.13, 1.83, and 10.16 nM) and capacity (Bmax = 1.30, 18.41, and 67.2 fmol/mg protein, respectively). Two classes of 125I-ANG II-binding sites of high affinity (KD = 0.11 and 1.76 nM) and low capacity (Bmax = 1.03 and 18.86 fmol/mg protein, respectively) were also identified. Cross-displacement studies confirmed that the two highest-affinity 125I-ANG III-binding sites and the 125I-ANG II-binding sites were the same. On the other hand, the binding of 125I-ANG III to the low-affinity 125I-ANG III-binding site could not be inhibited with ANG II. These data imply that previously measured differences in the biological potency of cerebroventricularly applied ANG III and ANG II probably do not result from differential binding of these peptides to central angiotensin receptors.

Angiotensin II↗
PubMed1988

The hypothalamic-angiotensin system: location and functional considerations.

Improved immunohistochemical and quantitative microiontophoretic methods were used to characterise angiotensinergic and angiotensin-sensitive neurones in the paraventricular nucleus (PVN) of the rat. The results can be summarised as follows: 1) Angiotensinogen was found in PVN neurones, astrocytes in the diencephalon which make putative contacts with microvessels, and in cells of the choroid plexus. 2) Affinity-purified angiotensin II/III antibodies were used to locate immunoreactive AII/III in large PVN neurones and their fibre tracts which project either caudally or ventrally to the neurohypophysis. 3) Quantitative microiontophoretic studies showed that PVN neurones are more sensitive to angiotensin II than to angiotensin II. 4) Iontophoretic co-application of the selective aminopeptidase inhibitors bestatin and amastatin, together with angiotensin II and angiotensin III produced results consistent with a central role for angiotensin III.

Aminopeptidases↗
PubMed1988

Metabolism of angiotensins II and III by membrane-bound peptidases from rat brain.

This study examines the metabolism of 125I-angiotensin II (125I-ANG II) and 125I-angiotensin III (applied 125I-ANG III or 125I-ANG IIIapp) by membrane peptidases. The first step in the metabolism of 125I-ANG II was the formation of 125I-ANG III (generated 125I-ANG III or 125I-ANG IIIgen). The ability of both ANG II and ANG III to reduce 125I-ANG IIIgen production without ANG(1-5), ANG(2-5), amastatin or bestatin being similarly effective suggests that this step may be highly substrate-specific. Subsequent metabolism of 125I-ANG IIIgen was similar to that of 125I-ANG IIIapp in that 125I-ANG(2-7) and 125I-ANG(2-4) fragments were produced. The formation of 125I-ANG(2-7) appeared to be a very substrate-specific process because it was only inhibited by ANG II and ANG III. In contrast, the production of 125I-ANG(2-4) was unaffected at the concentration of inhibitors used and is considered to be a relatively nonspecific process. However, despite these similarities sequential N-terminal cleavage leading to the formation of 125I-ANG(3-8), 125I-ANG(4-8) and 125I-Tyrosine appears to be the preferred pathway in the metabolism of 125I-ANG IIIapp. The absence of this pathway in the metabolism of 125I-ANG IIIgen suggests that applied and generated 125I-ANG III may be metabolized in separate degradative compartments. These data demonstrate that 125I-ANG II and 125I-ANG III are metabolized by membrane-bound peptidases in an orderly and sequential manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1987

Improved immunohistochemical staining of angiotensin II in rat brain using affinity purified antibodies.

Recent immunohistochemical studies that have sought to detect angiotensin II/III (AII/AIII) immunoreactive material in the brain have been forced to rely on a small number of antisera because most AII/AIII antibodies have unexplainably proved unsuitable for immunohistochemistry. Although extremely useful tools, these antisera have suffered from high background staining. The purpose of this study was to re-examine and characterize the staining using the most popular AII/AIII antiserum (Denise) before and after purification on an AII CH-sepharose affinity column. The use of crude AII/AIII antiserum resulted in the staining of large varicosities and cell bodies. Fibres were all but invisible owing to extensive background staining. In contrast, the purified antibodies yielded little background staining and produced a discrete staining of AII/AIII fibres with small varicosities in the paraventricular-hypophysial pathway and of cell bodies of large hypothalamic neurones. In addition punctate staining demarcated the perikarya of some neurones and resembled boutons containing immunoreactive AII/AIII. Biochemical and histochemical analysis of the crude antiserum, the affinity purified antibodies and other fractions off the sepharose column demonstrated that a large portion of the total staining (various types of background) seen with crude antiserum and column fractions was not to AII/AIII or several angiotensin-derived fragments. Furthermore, successful preabsorption blanks for the purified antibodies could only be achieved with AII coupled through its N-terminal, suggesting that these purified antibodies reacted best with conjugated angiotensin in the fixed tissue. In total the results of this study indicate that the background staining seen with crude antiserum is not to AII/AIII. The use of affinity purified antibodies greatly enhances resolution, enabling one to visualise even small fibres in rats not treated with colchicine, and should improve our ability to develop accurate maps of central angiotensinergic pathways.

Angiotensin II↗
PubMed1987

The effects of the aminopeptidase inhibitors amastatin and bestatin on angiotensin-evoked neuronal activity in rat brain.

During a recent comparison of iontophoretically applied angiotensin II (AII) and angiotensin III (AIII) in the paraventricular nucleus of the rat, we observed that the response latency for AIII was much shorter than that for AII. This suggested that AII may have to be converted to AIII before it becomes active. To test this hypothesis we performed 3 experiments. (1) We examined the effects of bestatin, an aminopeptidase B inhibitor, on the activity of applied AII and AIII. (2) Next, we monitored the effects of amastatin, a specific aminopeptidase A inhibitor, on the action of co-applied AII or AIII. (3) And, finally, we examined the response to the aminopeptidase-resistant analog Sar1-AII, both applied alone and in combination with AII or AIII. Bestatin, while having no activity of its own, dramatically enhanced the actions of both AII and AIII. Amastatin, on the other hand, had little effect on AII's action and diminished or totally blocked AII-dependent activity. Like bestatin, amastatin had no effect alone. Sar1-AII reduced spontaneous activity of angiotensin-sensitive neurons and inhibited the actions of AII and AIII in a reversible manner. The same cells were also blocked by the recognized angiotensin antagonist Sar1, Ile8-AII. In total these results strongly support the notion that AII must be converted to AIII in the brain before it is activated.

Aminopeptidases↗
PubMed1987

Heightened blood pressure and drinking responsiveness to intracerebroventricularly applied angiotensins in the spontaneously hypertensive rat.

The effects of bolus intracerebroventricular (i.c.v.) injections of angiotensin II (AII) and angiotensin III (AIII) on blood pressure and water consumption were investigated in Okamoto-Aoki spontaneously hypertensive rats (SHR), and Wistar-Kyoto (WKY) and Sprague-Dawley (SD) normotensive controls. Heightened sensitivity to i.c.v. administered AII and AIII was observed in the SHR as compared with WKY and SD strains for both pressor and drinking responses. The results are consistent with the notion that the SHR has a genetic defect that directly perturbs central angiotensinergic transmission. Two types of defects appear plausible, an alteration in the central angiotensin receptor and its associated transduction system and/or a decrease in the efficiency of signal termination. The present results are interpreted to primarily support the second possibility that a dysfunction in central aminopeptidase activity results in an extended life expectancy of angiotensin, and perhaps other peptides, that contribute to the hypersensitivity seen in the SHR.

Angiotensin II↗
PubMed1987

Reduction of 125I-angiotensin II binding sites in rat brain following monosodium glutamate treatment.

Adult Sprague-Dawley rats were pretreated with 6 g/kg of monosodium glutamate (MSG). After one month, specific binding of 125I-angiotensin II was measured in membrane preparations from the brain. A 40-45% decrease in the number of 125I-angiotensin II binding sites was observed in brain tissues containing circumventricular organs. No decrease in binding sites was observed in the thalamus/hypothalamus brain region, which resides totally within the blood-brain barrier. The results suggest that MSG can destroy angiotensin-sensitive neurons in circumventricular structures, which are located primarily outside the blood-brain barrier.

Angiotensin II↗
PubMed1987

Localization of angiotensinogen in multiple cell types of rat brain.

Angiotensinogen was localized in 3 cell types in brain using immunohistochemical methods. These locations included subpopulations of neurons in nuclei that co-stain for angiotensin II, subpopulations of astrocytes that make putative contacts with brain microvessels, and cells of the choroid plexus. These findings are consistent with multiple functions for brain angiotensinogen as a precursor for neuronal angiotensin II and as a potential source for angiotensin II that is locally produced in the brain.

Angiotensin II↗