Biomedical subjects

J W Harding

Publications and source records attributed to J W Harding.

At least 73 records · Page 4Linked to original sources

PubMed1992

Release of angiotensins in paraventricular nucleus of rat in response to physiological and chemical stimuli.

The brain angiotensin (ANG II and III) system is known to play an important role in the central control of cardiovascular function and body water homeostasis. A number of components of the angiotensin system including active peptides, precursors, synthetic enzymes, and receptors have been localized to specific brain nuclei including the paraventricular nucleus (PVN) of the hypothalamus. We and others have hypothesized that the PVN is a major integrative hub of the central angiotensin system receiving angiotensinergic input from central detectors (circumventricular organs) and sending efferents to higher brain and spinal cord centers. Implicit in this idea is that angiotensins, like all neurotransmitters, should be releasable with appropriate chemical and physiological stimuli. Therefore we examined the ability of water deprivation or direct infusion of either 65 mM K+ or 80 microM veratridine to stimulate the release of angiotensins from the PVN of the rat. Using push-pull cannulas to perfuse the PVN and radioimmunoassay (RIA) to analyze the superfusate for immunoreactive angiotensins, we established that 24 h of water deprivation resulted in an approximate 5-fold increase in the angiotensin release rate, whereas 48-h deprivation produced a dramatic 492-fold increase in release. Direct infusion of 65 mM K+ into the PVN was unable to stimulate angiotensin release, but 80 microM veratridine elicited a sevenfold increase in the angiotensin release rate. High-performance liquid chromatographic separation and RIA analysis of veratridine- and water deprivation-stimulated angiotensin release demonstrated that 93.4% of the releasable angiotensin coeluted with ANG III, whereas only 6.8% eluted with authentic ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗

PubMed1992

Role of paraventricular nucleus in control of blood pressure and drinking in rats.

The present investigation examined the abilities of angiotensin (ANG) II and III to produce increases in blood pressure and drinking when microinfused into the paraventricular nucleus (PVN) of the hypothalamus of the Sprague-Dawley rat. Dose-dependent elevations in systemic blood pressure and heart rate were measured to both ANG II and III in the anesthetized rat, with ANG II more potent than ANG III at the two highest doses examined. Pretreatment with the specific ANG receptor antagonist [Sar1,Thr8]ANG II (sarthran), blocked subsequent ANG II- and III-induced elevations in blood pressure, suggesting that these responses were dependent on the activation of ANG receptors. A similar analysis in awake rats yielded nearly equivalent results. A final experiment demonstrated that microinfusions of ANG II and III into the PVN produced drinking in a dose-dependent manner, with greater consumption to ANG II than ANG III. Again, sarthran was found to block the dipsogenic response. Histological examination revealed that the location of the injection site was linked to the character of the ANG-dependent response. These data suggest that the PVN may play a critical role in mediating central ANG effects on body water homeostasis and blood pressure regulation. Furthermore, it appears that subnuclei of the PVN may participate differentially in ANG-mediated actions.

Angiotensin II↗

PubMed1991

The AT2 angiotensin receptor subtype predominates in the 18 day gestation fetal rat brain.

The angiotensin II receptor subtype-specific antagonists Dup 753 (AT1) and PD 123177 (AT2) were used to characterize the angiotensin II receptor subtypes present in 18 day gestation fetal Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rat brain using in vitro receptor autoradiography. The AT2 subtype was predominant in the brain of both rat strains, even in areas that display predominantly the AT1 subtype in the adult rat brain.

Angiotensin II↗

PubMed1991

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas.

Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar1,Ile8-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTP gamma S. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.

Actins↗

PubMed1991

Hypotensive effects of sarthran in normotensive and spontaneously hypertensive rat strains.

The specific angiotensin receptor antagonist [Sar1, Thr8]AII (sarthran) was intracerebroventricularly (ICV) infused in anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) normotensive controls. The results extend earlier findings by determining that: 1) the hypotensive effect of ICV-infused sarthran could be enhanced in anesthetized as compared with alert animals; 2) SHRs revealed a greater hypotensive response as compared with WKY rats; and 3) no sarthran-induced agonistic effects were observed in contrast with previous results using alert SHRs. These findings support the use of sarthran as a potent angiotensin receptor antagonist to investigate the role of the brain angiotensin system in the control of normal and dysfunctional blood pressure.

Angiotensin II↗

PubMed1991

Use of aminopeptidase M as a hypotensive agent in spontaneously hypertensive rats.

The present investigation determined that a commercially available aminopeptidase M (AmM, Sigma Chemical) can be utilized to lower blood pressure in normotensive and hypertensive rats. In vitro analyses indicated that the predominant peptidase present in this preparation was AmM; however, it also contained some aminopeptidase A (AmA) and less DAP IV. Although no DAP IV-mediated metabolism of angiotensin II (AII) or angiotensin III (AIII) was measured, both AmM and AmA metabolized AII and AIII. Upon further examination, it appeared that AII could be converted to AIII by either AmM or AmA; however, Arg was cleaved from the N-Terminal of AIII predominantly by AmM. The aminopeptidase inhibitors actinonin (AC), amastatin (AM), and bestatin (BE) effectively blocked the AmM-induced hydrolysis of the Asp-Arg bond of AII, and the Arg-Val bond of AIII. The activity of AmA was inhibited by AM but was relatively resistant to inhibition by AC and BE. Next, exogenous aminopeptidase replacement was employed in the anesthetized spontaneously hypertensive rat (SHR) in an attempt to temporarily correct a hypothesized brain deficiency of receptor-associated peptidases and lower blood pressure. Third-ventricle infusion of AmM produced significant drops in blood pressure and heart rate in both SHRs and Wistar-Kyoto normotensive controls. Pretreatment with AC or BE was particularly effective at interfering with the subsequent AmM-induced hypotensive effect, while AM was less effective. The central mechanisms underlying these effects are in need of further investigation; however, they are at least partially dependent upon the brain angiotensin system.

Aminopeptidases↗

PubMed1991

Solubilization and partial characterization of angiotensin II receptors from rat brain.

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.

Angiotensin II↗

PubMed1991

Endothelin binding in brain of normotensive and spontaneously hypertensive rats.

The endothelins (ETs) are a recently discovered family of peptides which appear to be involved in hemodynamic regulation; they have potent vasoconstrictor properties and dose-related effects on blood pressure when administered peripherally. Little is known about the role of ET in the brain. The purpose of this study was to characterize the binding properties of various ETs in the brain of normotensive (Wistar-Kyoto) and hypertensive (spontaneously hypertensive) rats. [125I]ET 1 was prepared using the enzymobead lactoperoxidase method and purified by high-pressure liquid chromatography. Membrane fractions were prepared from homogenates of various brain regions. A differential distribution of ET binding was found among the 14 brain regions studied. The cerebellum, brainstem and area postrema/nucleus tractus solitarius had the highest binding, whereas the cortex, pituitary and septum had the least binding. Competition experiments performed with hypothalamus, brainstem and cerebellum demonstrated different Ki values for the ET studied. ET 2 had the highest affinity with a Ki of 4 x 10(-1) M, whereas the ET analog, Ala3,11-ET 1, had the lowest affinity with a Ki of 3 x 10(-10) M. Saturation experiments indicated a single class of high-affinity receptors in cerebellar (Kd = 2.5 x 10(-11) M, maximal binding Bmax = 1.25 x 10(-12) mol/mg) and hypothalamic membranes (Kd = 1.9 x 10(-11) M, Bmax = 0.93 x 10(-12) mol/mg). No differences in Kd or Bmax were detected between Wistar-Kyoto and spontaneously hypertensive rats hypothalamic and cerebellar tissues. The results of this study suggest a role for ET in the brain, but revealed no differences between normotensive and hypertensive strains.

Angiotensin II↗

PubMed1990

Intracerebroventricularly applied peptidase inhibitors increase endogenous angiotensin levels.

Rats received the aminopeptidase inhibitors amastatin (AM) and bestatin (BE), and carboxypeptidase inhibitor Plummer's (PL) via intracerebroventricular infusion in various combinations, i.e. PL alone, AM + BE, and a cocktail consisting of AM + BE + PL. Blood pressure responses were recorded and a postinfusion sample of cerebrospinal fluid (CSF) was radioimmunoassayed for endogenous angiotensin levels. Results indicate that CSF angiotensin was increased approximately 1.5x over control levels when PL was infused; a 2.5x increase accompanied AM + BE administration; and a 10.3x elevation was measured when all 3 inhibitors were infused as a cocktail. Concomitant elevations in blood pressure accompanied increased concentrations of angiotensin. We conclude that endogenous levels of angiotensin can be significantly increased in the ventricular space when a combination of these inhibitors is utilized to protect both the amino and carboxyl terminals of the angiotensin molecule from enzymatic degradation.

3-Mercaptopropionic Acid↗

PubMed1990

Comparison of angiotensin metabolism by brain membranes from SHR and WKY rats.

The ability of membrane-associated peptidases from the brains of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats to metabolize iodinated angiotensin (125I-Ang II) and 125I-Ang III was compared. 125I-Ang II was metabolized to 125I-Ang III and other fragments exclusively by membrane-associated peptidases. In contrast to 125I-Ang III which was effectively degraded by both membrane-associated and residual cytosolic peptidases, 125I-Ang II was unaltered by contaminating cytosolic enzymes. The ability of SHR-derived membranes to metabolize 125I-Ang II and produce 125I-Ang III was enhanced when compared to membranes from WKY rats. No difference was observed in the ability of membrane or cytosolic enzymes from SHR and WKY rats to degrade 125I-Ang III. These data are consistent with an increased availability of Ang III in the brains of SHRs.

Angiotensin II↗

PubMed1990

Intracerebroventricularly infused [D-Arg1]angiotensin III, is superior to [D-Asp1]angiotensin II, as a pressor agent in rats.

Two D-amino acid substitution angiotensin analogues were compared against native angiotensin II (AII) and angiotensin III (AIII) for their resistance to brain tissue-induced degradation and for pressor potency when intracerebroventricularly (i.c.v.) infused in Sprague-Dawley rats. The in vitro results indicate that [D-Asp1]AII was very resistant to degradation, AII and [D-Arg1]AIII were degraded at similar rates, while AIII was the most rapidly degraded. In vivo results revealed that AII, AIII and [D-Arg1]AIII produced greater pressor responses than [D-Asp1]AII. Intracerebroventricular pretreatment with the aminopeptidase A inhibitor, amastatin, significantly reduced the subsequent pressor response to i.c.v. infused [D-Asp1]AII presumably by inhibiting its conversion to AIII. In contrast, pretreatment with the aminopeptidase B inhibitor, bestatin, potentiated the subsequent pressor response to i.c.v. infused [D-Arg1]AIII, presumably by inhibiting the conversion of [D-Arg1]AIII to the less active hexapeptide AII(3-8). Next, i.c.v. pretreatment with the specific angiotensin receptor antagonist, [Sar1, Thr8]AII (Sarthran) was found to greatly diminish the subsequent pressor responses to i.c.v. infused [D-Asp1]AII and [D-Arg1]AIII, suggesting that these analogues are having their effect at the same brain angiotensin receptor site. These results support the hypothesis that AIII, or AIII-like ligands, may serve as the active form of brain angiotensin.

Aminopeptidases↗

PubMed1990

Aminopeptidase-induced elevations and reductions in blood pressure in the spontaneously hypertensive rat.

In vitro results indicated that human placenta-derived aminopeptidase A (APA) was very effective at hydrolyzing aspartate from the angiotensin molecule, thus converting angiotensin II to angiotensin III, but was not active against angiotensin III. In vivo experiments revealed significant elevations in blood pressure when APA was intracerebroventricularly infused into anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive control rats (WKY), with maximum mean (+/- s.e.m.) increases of 30.0 +/- 2.5 and 32.5 +/- 3.7 mmHg, respectively. By contrast, in vitro incubation results utilizing leucine aminopeptidase M (LAP-M) indicated very active degradation of angiotensin III, with less rapid degradation of angiotensin II. The intracerebroventricular infusion of LAP-M significantly reduced blood pressure, particularly in the SHR, but also in WKY, -65.8 +/- 5.1 and -42.5 +/- 6.1 mmHg, respectively. Pretreatment with the specific angiotensin receptor antagonist, Sar1, Thr8 angiotensin II (sarthran) significantly diminished the subsequent APA-induced increase in blood pressure in members of both strains. Pretreatment with sarthran has previously been shown to significantly diminish LAP-M-induced decreases in blood pressure in SHR. Thus, the effects of these aminopeptidases appear to be primarily dependent upon the brain angiotensinergic system, and are consistent with the hypothesis that angiotensin III is the primary active form of central angiotensin.

Aminopeptidases↗

PubMed1990

Inherited lysozyme deficiency in rabbits. The absence of a primary isozyme of lysozyme as the cause of the condition.

Lysozyme from normal and genetically lysozyme-deficient rabbits was extracted from three types of tissue: leukocytic (bone marrow), lymphoepithelial (thymus and appendix), and gastrointestinal (colon). Extracts were analyzed by electrophoretic, chromatographic, and kinetic techniques. Identification of at least two isozymes of rabbit lysozyme was made with these techniques. The distribution of the isozymes was tissue specific. Leukocytic and gastrointestinal isozymes were clearly distinguished, and a possible lymphoepithelial isozyme that resembled the gastrointestinal isozyme electrophoretically and chromatographically but not kinetically was identified. Mutant, lysozyme-deficient rabbits lacked completely a detectable leukocytic isozyme but had gastrointestinal and lymphoepithelial isozyme(s) indistinguishable from those of normal rabbits. By electrophoretic methods, the mutant rabbits were demonstrated to lack a protein band corresponding to that of the leukocytic isozyme of lysozyme from normal rabbits. These observations considered collectively were interpreted as evidence that at least two primary isozymes of lysozyme are present in rabbits and that inherited lysozyme deficiency in rabbits is caused by the absence of a single primary isozyme.

Animals↗

PubMed1989

Increased blood pressure induced by central application of aminopeptidase inhibitors is angiotensinergic-dependent in normotensive and hypertensive rat strains.

Two aminopeptidase inhibitors, amastatin (AM) and bestatin (BE), were employed in 3 strains of rats, spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD), to investigate the central angiotensinergic system. The results indicate that intracerebroventricular (i.c.v.) injections of AM and BE induced pressor elevations in all 3 strains of rats. In order to test for the possibility of spillage into peripheral vasculature, members from all 3 strains were peripherally infused with AM, BE, or 0.15 NaCl via jugular vein catheters. The SHRs were significantly more responsive to the aminopeptidases than the normotensive strains, however their overall pressor responses were only 33% of those to i.c.v. infusion. Next, in order to test the notion that these aminopeptidase inhibitors are having their effect via the central angiotensinergic system, and not some other peptidergic system, the specific angiotensin receptor antagonist, Sar1, Thr8-AII (sarthran) was employed. Intracerebroventricular pretreatment with sarthran prevented subsequent pressor responses to i.c.v. AM and BE in members of all 3 strains, thereby suggesting that these aminopeptidase inhibitors are having their effect via the central angiotensinergic system.

Aminopeptidases↗

PubMed1989

Hypothalamic angiotensinergic fibre systems terminate in the neurohypophysis.

A new affinity-purified anti-angiotensin II/III antibody ('BODE') was used to determine the location of angiotensin-like immunoreactivity in the paraventriculo-hypophysial pathway, especially in the pituitary. Angiotensin-like immunoreactivity was shown to be concentrated in the neurohypophysis and was characterised by a dense plexus of fibres and terminals.

Angiotensin II↗

PubMed1989

Autoregulation of cochlear blood flow in normotensive and spontaneously hypertensive rats following intracerebroventricularly mediated adjustment of blood pressure.

Previous studies in our laboratory (Quirk et al., 1988) noted significantly impaired elevations in cochlear blood flow (CoBF) during systemic infusion of the potent vasoconstrictive agent angiotensin II (AII) in the spontaneously hypertensive rat (SHR) as compared with the normotensive Wistar-Kyoto (WKY) rat, despite similar increases in systemic blood pressure. We interpreted these results to suggest that SHRs have an exaggerated autoregulatory mechanism that controls blood supply to the cochlear vessels. However, an alternative explanation for these findings concerns the potential influence of the elevated baseline blood pressure of the SHR on CoBF. Specifically, if there is an absolute threshold blood pressure that triggers an autoregulatory response in the cochlea, then the SHRs would reach that threshold sooner than normotensive animals because they begin at a baseline blood pressure that is well above that of the WKY rat. The present study addressed this possibility by pharmacologically reducing SHR blood pressure to WKY baseline blood pressure and raising WKY to SHR baselines, followed by the infusion of previously utilized doses of AII. The results are consistent with previous findings and support our interpretation of an exaggerated autoregulation of cochlear blood supplying the SHR.

Angiotensin II↗

PubMed1989

Structure-function analyses of brain angiotensin control of pressor action in rats.

The present investigation examined the relative pressor potencies of intracerebroventricularly infused angiotensin (ANG) II, successively shortened COOH-terminal fragments through ANG II(5-8), and the analogues [Sar1]ANG II through [Sar1]ANG II(5-8). The results indicate that ANG II, ANG III, [Sar1]ANG II, and [Sar1]ANG III were identical with respect to pressor responses in the alert free-moving rat. In addition, ANG II(3-8) and [Sar1]ANG II(3-8) exhibited 68-70% of the activity of the above compounds, whereas the activity of the shorter COOH-terminal fragments dropped to approximately 13-35%. Pressor responses caused by each of the active forms of angiotensin could be substantially reduced by pretreatment with the specific angiotensin receptor antagonist [Sar1,Thr8]ANG II (Sarthran), suggesting either that these ligands are acting at multiple receptors for ANG II and its fragments, which are all blocked by Sarthran, or that the ligands are acting at a common receptor site. These results, coupled with other recent findings, suggest that the brain angiotensin receptor may be designed to preferentially interact with ANG II and/or ANG III or other angiotensin analogues that structurally resemble ANG III such as [Sar1]ANG II. It is concluded that ANG III's importance as a centrally active ligand has been underestimated and that ANG III may be an active form of angiotensin in the brain.

Amino Acid Sequence↗

PubMed1989

Leucine aminopeptidase M-induced reductions in blood pressure in spontaneously hypertensive rats.

Leucine aminopeptidase M significantly reduced blood pressure for up to 40 minutes when infused intracerebroventricularly into anesthetized spontaneously hypertensive rats (SHR) from a mean +/- SEM of 190 +/- 4 to 94 +/- 7 mm Hg and also in normotensive Wistar-Kyoto (WKY) rats from 138 +/- 5 to 68 +/- 8 mm Hg. Cerebrospinal fluid levels of angiotensin II (Ang II) and III were measured by radioimmunoassay and indicated drops with leucine aminopeptidase M infusion in SHR (from 36 +/- 6 to 11 +/- 1 pg/100 microliters) and in WKY rats (from 33 +/- 9 to 13 +/- 1 pg/100 microliters). Pretreatment with the specific angiotensin receptor antagonist [Sar1, Thr8]Ang II (sarthran) significantly diminished the subsequent leucine aminopeptidase M-induced decreases in blood pressure in SHR and facilitated recovery to base level blood pressure and heart rate in blood strains. Thus, exogenous application of leucine aminopeptidase M into the brain lateral ventricles of SHR is temporarily effective at reducing blood pressure, and this effect appears dependent on the brain angiotensinergic system. This treatment also reduced blood pressure in WKY rats; however, pretreatment with sarthran was reasonably ineffective at preventing subsequent leucine aminopeptidase M-induced decreases in blood pressure.

Angiotensin II↗