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J W Harding

Publications and source records attributed to J W Harding.

At least 109 records · Page 6Linked to original sources
PubMed1987

Angiotensin-sensitive neurons in the rat paraventricular nucleus: relative potencies of angiotensin II and angiotensin III.

Angiotensin-activated neurons were examined using microiontophoretic methods in the paraventricular nucleus (PNV) of the rat. In all cases angiotensin III (AIII) was more potent than angiotensin II (AII). This greater sensitivity to AIII was manifested by lower thresholds, shorter latencies, and higher spike frequencies/amplitudes of applied current. The superior potency of AIII was further exaggerated in the spontaneously hypertensive rat (SHR) compared with normotensive Wistar Kyoto (WKY) rats. Postactivity for both AII and AIII was greatly prolonged in SHR. This appeared specific since no prolongation in acetylcholine postactivity was seen in SHR. These data support the notion that AIII may be the centrally active form of angiotensin and are consistent with an obligatory conversion of AII to AIII prior to activation. The selective enhancement of postactivity observed in SHR following angiotensin application suggests a possible defect in signal termination.

Angiotensin II↗
PubMed1987

Amastatin and bestatin-induced dipsogenicity in the Sprague-Dawley rat.

Intracerebroventricular application of the aminopeptidase inhibitor bestatin, but not amastatin, demonstrated a dose-dependent drinking response. Amastatin is a selective, but not totally specific aminopeptidase inhibitor that blocks aminopeptidase A, which cleaves acidic amino acids, while bestatin selectively blocks aminopeptidase B, which cleaves basic amino acids. Thus, amastatin's major action should be to inhibit angiotensin II (AII) to angiotensin III (AIII) conversion and bestatin's to block AIII degradation. Treatment with the angiotensin receptor antagonist, Sar, Thr-AII (sarthran), completely inhibited bestatin-induced drinking at two different doses. These results support a critical role for the brain-angiotensin system in the ongoing regulation of body fluid homeostasis and suggest an important role for angiotensin III in the brain.

Aminopeptidases↗
PubMed1987

Quantification of angiotensin iontophoresis.

A method of value for studying the effects of angiotensin II (AII) and angiotensin III (AIII) on the brain is microiontophoresis combined with single unit recording. The purpose of this study was to quantitate the release of angiotensins under various experimental conditions thus providing a firm basis for the iontophoretic application of angiotensins. Quantification of release was accomplished by adding the appropriate [3H]angiotensin to 1 X 10(-3) M solutions of AII and AIII and then measuring the counts released from the tip of the microiontophoretic pipette in vitro into a small volume of Ringer solution. Although both AII and AIII were released by diffusion from micropipettes, this release could all but be eliminated with a retaining current of 20 nA. The release of AII and AIII was linear with respect to the amount of ejecting current applied up to 60 nA, the highest current examined. Angiotensin II at pH 4.5 and AIII at pH 3.5 were released at similar rates of 41.5 and 45.1 fmol/min/nA respectively. Raising the pH of the AII solution to 4.5 reduced the rate of release to 17.9 fmol/min/nA. Transport numbers were determined as follows: AII pH 3.5-0.115; AII pH 4.5-0.035, and AIII pH 4.5-0.145. It can be concluded that angiotensins are readily released by microiontophoresis, the response is linear with respect to application current, and that with the use of the appropriate pH the rate of release of AII and AIII are comparable.

Angiotensin II↗
PubMed1987

Angiotensin's contribution to dipsogenic additivity in several rodent species.

Significant positive correlations were obtained between plasma angiotensin levels and drinking following the administration of polyethylene glycol (PEG) in rats, gerbils, and hamsters and following isoproterenol (ISOP) in rats and gerbils, but not in a South American rodent, Octodon degus (degus). Hamsters revealed elevations in plasma angiotensin following ISOP but no drinking, whereas degus failed to show changes in plasma angiotensin to either treatment, although drinking occurred following PEG. All species drank to 2.5 M NaCl injections with no measurable changes in plasma angiotensin concentrations. A second experiment addressed our previous inability to measure [125I] angiotensin II (AII) specific binding in the brains of gerbils and degus and indicated that these animals possess circumventricular organ (CVO) angiotensin III (AIII) receptors; thus, circulating AIII may be the ligand in these species. A final experiment examined members of these species for dipsogenic additivity following the pairing of extracellular and intracellular thirst challenges. Rats and gerbils revealed additivity when challenged with either PEG or ISOP paired with 2.5 M NaCl. Degus and hamsters indicated additivity only with PEG and 2.5 M NaCl combined. Despite the presence of CVO angiotensin receptors in degus and hamsters, it is concluded that the important component of the hypovolemic dipsogenic stimulus in members of these species may be activation of volume receptors rather than brain angiotensin receptors.

Angiotensin II↗
PubMed1987

Delayed cerebroventricular metabolism of [125I]angiotensins in the spontaneously hypertensive rat.

This study was designed to evaluate the hypothesis that impaired brain angiotensin signal termination contributes to the sustained blood pressure elevations noted in the genetically hypertensive rat model of human essential hypertension. A technique that combined the intracerebroventricular injection of [125I]angiotensins, followed by focused microwave fixation to stop all peptidase activity and subsequent HPLC analyses, was used for determining half-lives of [125I]angiotensin II and [125I]angiotensin III in the ventricular space. The results indicate that the spontaneously hypertensive rat evidenced significantly longer half-lives for intracerebroventricularly injected [125I]angiotensin II over those measured for the Wistar-Kyoto and Sprague-Dawley normotensive rat strains: 45.0, 27.2, and 25.0 s, respectively. This was also true for intracerebroventricularly administered [125I]angiotensin III: 19.5, 11.4, and 9.0 s, respectively. These results support the notion that a dysfunction in central aminopeptidase activity in the spontaneously hypertensive rat may result in prolonged half-lives of endogenously synthesized angiotensins II and III, which are known to serve as ligands at central angiotensin receptors responsible for the control of cardiovascular function. The extended half-lives of these ligands may contribute to the sustained elevations in blood pressure observed in this animal model.

Angiotensins↗
PubMed1987

Comparison of angiotensin II staining in rat brain using affinity purified and crude antisera.

The use of affinity purified ANG II antiserum as opposed to crude antiserum greatly enhanced the staining resolution in rat brain. This improved resolution was due to a complete loss of background staining and an apparent increase in specific staining that was totally blockable by preabsorption. With the purified antibody it was easily possible to visualise the finest fibres in rats not treated with colchicine. Furthermore, the improved technique permitted a clearer visualisation of an ANG II-like immunoreactive product in cell bodies. This use of affinity purified antibody should greatly facilitate the mapping of central angiotensinergic pathways.

Angiotensin II↗
PubMed1987

Binding, degradation and pressor activity of angiotensins II and III after aminopeptidase inhibition with amastatin and bestatin.

In the metabolism of angiotensin peptides by tissue angiotensinases, aminopeptidases A, B, M and leucine aminopeptidase have been identified as being particularly effective. Because the inhibitory actions of amastatin (AM) and bestatin (BE) are relatively specific for these aminopeptidases, we have examined the effects of these inhibitors on the binding, degradation and pressor activity of angiotensin II (AII) and angiotensin III (AIII). Within 30 min at 37 degrees C, significant metabolism of 125I-AII and 125I-AIII by homogenates of a block of tissue containing hypothalamus, thalamus, septum and anteroventral third ventricle regions of the brain was observed. A majority of 125I-AIII metabolism was due to soluble peptidases, whereas that of 125I-AII primarily resulted from membrane-bound peptidases. AM, BE and reduced incubation temperatures significantly decreased the metabolism of 125I-AII and 125I-AIII. After appropriate adjustments to reflect the proportion of intact radioligand bound, temperature- or inhibitor-induced decreases in metabolism were matched by corresponding increases in specific binding. Heat-treated bovine serum albumin, as a nonspecific peptidase inhibitor, had no effect on either the metabolism or binding of the ligands used. In accordance with their actions in vitro, i.c.v. administration of AM and BE prolonged the pressor activity of subsequently applied AII and AIII. Unexpectedly, the amplitude of the pressor response to AIII was increased by BE, whereas that to AII was decreased by AM. The results of this study indicate that the metabolism of AII and AIII by aminopeptidases is relatively specific and acts to modulate the actions of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopeptidases↗
PubMed1986

High-performance liquid chromatographic analysis of 'specifically bound' label after [125I]angiotensin II binding to rat brain membranes.

The combined hypothalamus-thalamus-septum and anteroventral third ventricular region (HTSA) of the rat was examined for [125I]angiotensin II ([125I]AII) binding using two protocols: one that preserved synaptosomal structure and a second that did not. Although maximum binding (Bmax) and dissociation constants (Kd) were similar in both preparations, high-performance liquid chromatographic analyses revealed that [125I]AII made up the majority of specifically bound label in the synaptosome preserved preparation while [125I]tyrosine ([125I]Tyr) represented most of the specifically bound label in the disrupted preparation. These results indicate that [125I]Tyr accumulation occurred subsequent to binding and degradation of [125I]AII and are consistent with the notion that rapid internalization of the receptor-[125I]AII complex occurs in those preparations where the synaptosomal structure remains intact.

Angiotensin II↗
PubMed1986

Effect of intraventricular infusion of an angiotensin II antagonist on 125I-angiotensin II binding in rats.

The effects of chronic (six day) intracerebroventricular (i.c.v.) infusion of an angiotensin II antagonist, sarcosine1, isoleucine8 angiotensin II ([Sar1, Ile8]Ang II), (500 ng/microliter per hour) was studied. Specific 125I-Ang II binding site density and binding affinity in the hypothalamus-thalamus-septum-midbrain (H-T-S-M) region of the brain and the adrenal medulla did not differ significantly between [Sar1, Ile8]Ang II treated and control (0.9% saline) rats. However, 125I-Ang II binding to the adrenal cortex was significantly reduced by i.c.v. infusion of [Sar1, Ile8]Ang II. The drinking response to microinjection of Ang II was blunted for up to seven days of [Sar1, Ile8]Ang II infusion. Thus, although [Sar1, Ile8]Ang II effectively blocked the central Ang II receptors, chronic infusion of this Ang II antagonist did not appear to cause alterations in brain H-T-S-M Ang II receptors, suggesting that brain Ang II receptors in normal rats do not undergo homologous regulation.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1986

Different pharmacological anatomy in the paraventricular hypothalamic nucleus, supraoptic nucleus, and suprachiasmatic nucleus of rats: quantitative autoradiography on angiotensin II receptor binding sites.

Angiotensin II (AII) and vasopressin (VP) play important roles in cardiovascular function. Using 125I-[Sar1,Ile8]-angiotensin II (125I-SI-AII), a potent AII antagonist, AII receptor binding sites were autoradiographically localized in three VP-producing areas of the hypothalamus and compared in hypertensive and normotensive rats. Within three major VP-producing areas, AII receptor binding was highest in the paraventricular hypothalamic nucleus and lowest in the supraoptic nucleus, suggesting that a differential AII regulation of separate VP systems exists in the brainstem. No statistical difference in 125I-SI-AII receptor binding was found between WKY and SHR rats in each of the three major VP-producing nuclei studied. These results are consistent with a role of AII receptors in a subtle and complicated regulation of VP in cardiovascular function.

Angiotensin II↗
PubMed1986

Elevations in plasma angiotensin II with prolonged ethanol treatment in rats.

Chronic alcohol consumption frequently leads to hypertension in humans. While previous reports have implicated the renin-angiotensin system as a potential mediator of this effect, plasma angiotensin II (AII) levels were either not measured or yielded negative results. The present investigation noted significant elevations in circulating AII in rats intubated daily with ethanol (4 g/kg) for 50 days. Animals administered ethanol only once evidenced AII concentrations equivalent with water intubated controls. Radioligand binding assay data indicated no differences in the number or affinity of Sar1,Ile8-AII binding sites in the thalamus, septum-anterior ventral third ventrical region or adrenal gland comparing those groups chronically treated with ethanol to water intubated controls. These results may support a role for the vasoconstrictive hormone AII in the etiology of alcohol-induced hypertension.

Adrenal Glands↗
PubMed1986

Quantitative autoradiography of 125I-[Sar1, Ile8]-angiotensin II binding in the brain of spontaneously hypertensive rats.

The brain contains its own angiotensin II (AII) system. To better understand the role of central AII in cardiovascular regulation, we used 125I-[Sar1, Ile8]-AII (125I-SI-AII), radioactive AII antagonist, to autoradiographically localize putative AII receptor binding in many parts of the central nervous system of the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. With 125I-SI-AII binding on brain membrane preparations. Scatchard analysis indicated that Kd values were from 0.10 +/- 0.04 nM to 0.13 +/- 0.05 nM, whereas Bmax values (femtomol/mg protein) were found to be from 6.95 +/- 1.60 to 15.52 +/- 4.99 among brain regions studied. Various SI-AII receptor binding activities among brain regions revealed in this study were therefore most likely due to differences in AII receptor density with high affinity binding of 125I-AII. Using 125I-SI-AII, specific binding for SI-AII was found in the nucleus tractus solitarius (NTS), paraventricular hypothalamic nucleus (PVN), subfornical organ (SFO), suprachiasmatic nucleus (SCN), area postrema, the dorsal motor nucleus of the vagus (DMX), and the nucleus of spinal tract of the trigeminal system (NSV). With quantitative receptor autoradiography in conjunction with radioactive standards, we have observed that the NTS possesses the highest SI-AII binding, followed by the PVN, SFO, NTS, DMX, and NSV. No significant differences were observed between the SHR and WKY rats in the SI-AII binding within the SFO, PVN and NTS. However, SHR at early hypertensive (7 weeks) and established hypertensive (16 weeks) stages contained significantly higher SI-AII bindings in the NSV, as compared to age-matched WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1986

Cerebroventricular and intravascular metabolism of [125I]angiotensins in rat.

This study compared the metabolism of [125I]angiotensin II (AII), [125I]angiotensin III (AIII), and [125I]Sar1,Ile8-AII (SI-AII) in the vascular and cerebroventricular compartments. Using HPLC methods to monitor degradation the following t1/2 values were established in the vascular compartment: AII, 12.7 +/- 1.4 s; AIII, 16.3 +/- 0.7 s; and SI-AII, 100.7 +/- 7.3 s. HPLC analysis also revealed that [125I]AII is converted in an obligatory manner to [125I]AIII during its degradation sequence. Cerebrospinal fluid contained no degradative capacity for [125I]AII but exhibited a significant capacity to degrade [125I]AIII. A technique that combined the intra-cerebroventricular injection of [125I]angiotensins followed by focused microwave fixation to stop all peptidase activity was used to determine the half-life of [125I]angiotensins in the ventricular space. Results indicated very rapid metabolism of angiotensins with the following t1/2 values: AII, 23.0 s; and AIII, 7.7 s. This extremely rapid, differential, and sequential metabolism of AII and AIII in two relevant body fluid compartments underscores the need for caution when interpreting data derived from intravascular and intracerebroventricular application of angiotensins. In addition the faster metabolism of AIII than AII in the ventricular space indicates that the actual potency of AIII at central angiotensin receptors is being underestimated.

Angiotensin II↗
PubMed1986

Manipulation of aminopeptidase activities: differential effects on iontophoretically applied angiotensins in rat brain.

During a recent comparison of iontophoretically applied angiotensin II (ANG II) and angiotensin III (ANG III) in the paraventricular nucleus of the rat we observed that ANG III was more potent than ANG II. This suggested that ANG II may have to be converted to ANG III before it becomes active. To test this hypothesis we performed two experiments. Firstly, we examined the effects of bestatin, an aminopeptidase B inhibitor, on the activity of applied ANG II and ANG III. Next, we monitored the effects of amastatin, a specific aminopeptidase A inhibitor, on the action of coapplied ANG II or ANG III. Bestatin, while having no activity of its own, dramatically enhanced the actions of both ANG II and ANG III. Amastatin, on the other hand, had little effect on ANG III's action and diminished or totally blocked ANG II-dependent activity. Like bestatin, amastatin had no effects alone. In total these results strongly support the notion that ANG II must be converted to ANG III in the brain before it is activated.

Aminopeptidases↗
PubMed1986

Heightened pressor effect and dipsogenicity to intracerebroventricularly applied angiotensin II and III in spontaneously hypertensive rats.

The effect of acute intracerebroventricular (i.c.v.) injections of angiotensin II and III (ANG II and ANG III; 0, 1, 10 and 100 pmol in 2 microliters artificial cerebrospinal fluid (CSF) on blood pressure and water consumption was investigated in Okamoto-Aoki spontaneously hypertensive rats (SHR), and Wistar-Kyoto (WKY) and Sprague-Dawley (SD) normotensive controls. Heightened sensitivity to i.c.v. ANG II and ANG III was observed in the SHR compared with the WKY and SD strains (P less than 0.001), for both pressor and drinking responses. In addition, i.c.v. treatment with an aminopeptidase B inhibitor, bestatin (20 nmol in 1 microliter artificial CSF) significantly potentiated the heightened pressor response to i.c.v.-injected ANG II and ANG III (100 pmol) in SHR and to a lesser degree in WKY animals compared with SD controls (P less than 0.001). These results suggest that a dysfunction in central aminopeptidase activity results in an extended life of endogenous angiotensins, and perhaps other peptides that may contribute to the high blood pressure seen in this animal model of human essential hypertension.

Angiotensin II↗
PubMed1985

Brain angiotensin II and III binding and dipsogenicity in the rabbit.

The rabbit brain evidenced low or non-detectable levels of [125I]angiotensin II binding but considerable [125I]angiotensin III specific binding in agreement with distributions reported for the primate brain. The rabbit was dispsogenically responsive to the intracerebroventricular infusions of angiotensin II and III; however, no elevations in blood pressure were measured for either peptide. Thus, members of this species appear to have limited usefulness as a primate model for central angiotensin-induced cardiovascular changes.

Angiotensin II↗
PubMed1985

Dysfunction of central angiotensinergic aminopeptidase activity in spontaneously hypertensive rats.

Alert spontaneously hypertensive (SH) rats, prepared with indwelling carotid artery catheters, demonstrated heightened and prolonged blood pressure (BP) responses to intracerebroventricular (i.c.v.) injections of 10 and 100 pmol angiotensin II and III (AII and AIII) as compared with Wistar-Kyoto (WKY) and Sprague-Dawley normotensive animals. Pretreatment with the aminopeptidase B inhibitor bestatin (10 nmol, i.c.v.) potentiated and prolonged the heightened pressor response to AIII (100 pmol, i.c.v.) in SH rats. These results suggest that dysfunction of angiotensin peptidase activity may be contributing to the progressive and sustained elevations in blood pressure noted to occur in the SH rat model of human essential hypertension.

Angiotensin II↗