Biomedical subjects

M I Phillips

Publications and source records attributed to M I Phillips.

At least 109 records · Page 6Linked to original sources

PubMed1990

Stimulation of phosphoinositide hydrolysis by oxytocin in renal epithelial cells.

Recently, it has been reported that oxytocin (OT) produces diuresis by its interaction with OT receptors in the kidney. LLC-PK1 cells have been used as a model system for renal epithelial cells. To determine if OT stimulates receptor-mediated phosphoinositide (PI) hydrolysis in LLC-PK1 cells as it does in nonrenal cell systems, we measured the release of PI hydrolysis products in LLC-PK1 cells by OT and a selective OT agonist (AK-2-60) in the absence and presence of a selective OT antagonist (KB-5-21). In addition, we determined the effect of an increase in osmolality of the incubation medium on OT-stimulated PI hydrolysis in LLC-PK1 cells. The methods involved the incubation of LLC-PK1 cells with [3H]inositol for its incorporation into membrane PI and the measurement of the release of [3H]inositol phosphates in the presence of LiCl which prevents dephosphorylation. The osmolality of the incubation media was increased from 300 mOsmol/kg of H2O to 600, 900 and 1200 mOsmol/kg of H2O by addition of NaCl and urea. In an iso-osmotic incubation medium OT (10(-11) M) produced a greater than 100% increase in PI hydrolysis in LLC-PK1 cells. The OT agonist, AK-2-60, produced a significant increase in PI hydrolysis in LLC-PK1 cells at 10(-8) M concentration. The effects of both OT and its agonist were concentration-dependent and were blocked by the OT antagonist, KB-5-21. An increase in osmolality of the incubation media decreased OT-stimulated PI hydrolysis in LLC-PK1 and abolished completely the effect of OT at 1200 mOsmol/kg of H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Dose-response testing of peptides by hippocampal brain slice recording.

The brain slice chamber described offers a method of studying, with intracellular electrodes, the relationship of response to dose of peptides. By raising the level of the slices 1 mm above the level of flowing perfusion medium, we can test substances in known concentrations, free from artifacts, during long duration, stable intracellular recordings. Manipulation of Ca2+/Mg2+ ratios in the medium can help to define synaptic and second messenger mediation of the responses. The addition of substances to the perfusion medium in this system could be combined with iontophoresis and/or micropressure techniques. Pathways in the slices may also be stimulated electrically and analyzed for the involvement of various synaptic transmitters. The results with the method so far show distinct differences among the peptides studied. Thus, there are several advantages to this method in establishing the physiological role of peptides in the brain.

Angiotensin II↗

PubMed1989

Chromatographic methods for characterization of angiotensin in brain tissue.

Ang II antiserum with high sensitivity and specificity was produced. The native Ang II antiserum was purified by affinity chromatography on Affi-Gel 102 with covalently coupled [Ile5]Ang II, and purified Ang II antiserum was covalently coupled to Affi-Gel 10. The column with the covalently coupled Ang II antiserum was used for the specific enrichment of Ang II from brain extracts. The efficiency and usefulness of affinity chromatography for the purification of Ang II from biological sources were tested with 125I-labeled, 3H-labeled, and synthetic [Ile5]Ang II added to rat brains prior to extraction. In addition, the methodology was used for the purification of endogenous Ang II from pig brain. The described three-step procedure for the isolation and purification of Ang II including extraction, affinity chromatography, and HPLC is rapid and highly specific with high loading capacity. We have applied the method to the peptide Ang II in brain, but the methodology may also be used in general for the rapid purification of other neuropeptides. A combination of HPLC with specific radioimmunoassays for Ang I and Ang II was utilized to demonstrate that rat brain cells in culture devoid of the influence of the peripheral RAS were able to synthesize radioactively labeled Ang I and Ang II after incubation with [3H]isoleucine. And, finally, an HPLC system capable of separating Ang I, Ang II, and its metabolites was used to obtain insight into the degradation pattern of angiotensin peptides in the brain. Aminopeptidases appear to be the major angiotensin-degrading enzymes, and endopeptidases do not appear to be involved.

Angiotensin II↗

PubMed1989

Autoradiographic analysis of alpha-adrenoceptors and muscarinic cholinergic receptors in the hyperplastic human prostate.

Radioligand receptor binding and autoradiography were used to characterize, localize and compare alpha-1 and alpha-2 adrenoceptors and muscarinic cholinergic receptor populations in human benign prostatic hyperplastic tissue. The binding of selective alpha-1 and alpha-2 ligands, [3H]-prazosin and [3H]-UK 14,304, to homogenates of human central and peripheral prostate was saturable and of high affinity. Scatchard analysis produced an equilibrium dissociation constant (KD) of 0.51 +/- 0.10 nM for alpha-1 adrenoceptors, and 2.34 +/- 0.40 nM for alpha-2 adrenoceptors. The mean densities, Bmax, of alpha-1 and alpha-2 adrenoceptors identified in the human adenomatous prostate were 65.9 +/- 12.9 and 36.1 +/- 7.0 fmoles/mg. protein respectively. Receptor autoradiography was used to examine the distribution of muscarinic cholinergic receptors [( 3H]-QNB), alpha-1 adrenoceptors [( 3H]-prazosin]), and alpha-2 adrenoceptors [( 3H]-rauwolscine) on consecutive sections of benign hyperplastic prostatic tissue. Although both subtypes of adrenoceptor were seen in the stromal component of the hyperplastic prostate, there was a substantial predominance of alpha-1 adrenoceptors. A densitometric computer-assisted analysis was performed on the autoradiographic slides to determine the mean ratio of specific alpha-1: alpha-2 adrenoceptors in the stromal compartment of the hyperplastic tissue. The ratio, expressed as % grain occupancy/unit area, was 3.9 +/- 0.75, which is in agreement with a functional alpha-1 adrenoceptor predominance shown in previous studies. Although sparsely distributed in the stroma, a dense alpha-2 adrenoceptor population was seen in association with blood vessels, and in close proximity to the base of some of the [3H]-QNB-labelled prostatic glandular epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1989

The captopril glomerular filtration rate renogram in renovascular hypertension.

Administration of captopril to animals with two-kidney, one clip, renovascular hypertension (RH) lowers the glomerular filtration rate (GFR) in the clipped kidney. The authors therefore tested the hypothesis that a decrease in GFR after captopril administration would identify patients with RH. Total GFR was measured by the plasma disappearance of Tc-99m-diethylenetriaminepentaacetic acid (DTPA) after bolus injection and single-kidney GFR from renal uptake of DTPA assessed by renography. The authors studied six patients with arteriosclerotic RH who had strongly lateralizing renal vein renin levels and greater than 80% stenosis of the renal artery to that kidney. Results were contrasted with those of six patients with essential hypertension (EH) with a similar mean arterial blood pressure (MABP). Captopril (50 mg orally) increased total GFR (ml/min) in all patients with EH (102 +/- 8 to 120 +/- 12, P less than 0.005). However, GFR decreased in patients with RH (73 +/- 8 to 61 +/- 9, P less than 0.05) after captopril. Although the single-kidney GFR of patients with RH decreased in all six stenotic kidneys (27 +/- 4 to 21 +/- 5, P less than 0.02), it did not change consistently in the contralateral kidneys (45 +/- 8 to 40 +/- 6, N.S.). Clonidine (0.3 mg) also lowered MABP in patients with RH but, unlike captopril, it did not reduce total kidney GFR (75 +/- 10 to 79 +/- 11, N.S.). In conclusion, short-term captopril administration increases GFR in patients with EH, but decreases it in those with RH. This action is unrelated to its depressor response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

Metabolism of angiotensin peptides by neuronal and glial cultures from rat brain.

The degradation pattern and rate of [Ile5]-Angiotensin (Ang) I, II, and III were studied in neuron-enriched and glia-enriched cells in primary cultures from rat brain. Metabolites were separated by HPLC, and their identities were evaluated by comparison of their retention times with those of synthetic Ang peptide fragments and by analysis of their amino acid composition. Major metabolites were identified as des-Asp1-[Ile5]-Ang I, des-Asp1-[Ile5]-Ang II, [Ile5]-Ang II (3-8) hexapeptide, [Ile5]-Ang II (4-8) pentapeptide, and [Ile5]-Ang II (5-8) tetrapeptide. Glia-enriched cells degraded [Ile5]-Ang I and [Ile5]-Ang III significantly faster than neuron-enriched cells, whereas no difference between the two types of cells was found in the degradation rate of [Ile5]-Ang II. Although the half-lives of [Ile5]-Ang I and [Ile5]-Ang III in neuron-enriched cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were not significantly different, neuron-enriched cultures from WKY rats metabolized [Ile5]-Ang II about 2.6 times faster than neuron-enriched cells derived from SHR.

Angiotensin I↗

PubMed1989

Alpha 2-adrenergic receptors in neuronal and glial cultures: characterization and comparison.

Membranes prepared from either neuronal or glial cultures contain alpha 2-adrenergic receptors as determined by the characteristics of [3H]yohimbine [( 3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 +/- 1.35 nM (n = 10) for neuronal cultures and 18.42 +/- 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a Bmax of 1.6 +/- 0.33 pmol/mg protein (n = 10) compared with 0.143 +/- 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for alpha 2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the alpha 2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynanthine, or propranolol. The agonists showed the same pattern with the alpha 2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog. 5'-guanylyl-imidodiphosphate, were able to lower the affinity of the alpha 2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of alpha 2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain alpha 2-adrenoceptors.

Animals↗

PubMed1989

Release of immunoreactive angiotensin II from neuronal cultures: adrenergic influences.

The effects of adrenergic drugs on the release of immunoreactive angiotensin II (ANG II-ir) from brain cells in culture were examined. In neuronal cultures, basal release of Ang II-ir was 43.65 +/- 7.44 pg/5-min incubation period (n = 14 experiments; 52 individual determinations), and in astrocytic glial cultures, it was 21.76 +/- 5.7 pg (n = 8 experiments; 24 individual determinations) when cells were exposed to buffer alone. Incubation of neuronal cultures with the alpha 2-adrenergic antagonist yohimbine (0.1-50 microM, 5 min) caused concentration-dependent increases in ANG II-ir release above basal levels. Analysis of the released material by high-pressure liquid chromatography revealed that authentic ANG II was present. No increase in the release of ANG II-ir was seen from glial cells. Experiments using neuronal cultures revealed that the yohimbine-induced release of ANG II-ir may be secondary to increased norepinephrine (NE) release. Incubation of neuronal cultures with NE (10 nM-50 microM) caused concentration-dependent increases in the release of ANG II-ir. This effect of NE was not inhibited by the alpha 1-adrenergic blocker prazosin. However, a weaker release of ANG II-ir from neuronal cultures was stimulated by the beta-adrenergic agonist isoproterenol at 100 microM. These data show that ANG II-ir can be released from neuronal but not glial cell cultures by adrenergic receptor-mediated mechanisms.

Adrenergic alpha-Agonists↗

PubMed1989

Effect of vagotomy on brain and plasma atrial natriuretic peptide during hemorrhage.

These experiments investigated the change in brain, atrial, and plasma concentrations of atrial natriuretic polypeptide (ANP) after a 33% hemorrhage and the role of vagal nerve input in these changes. In rats, hemorrhage decreased plasma ANP from 246 +/- 48 to 41 +/- 7 pg/ml, but in a hypothalamic tissue block ANP increased from 19.0 +/- 0.9 to 25.5 +/- 0.6 ng/g tissue (P less than 0.05). Bilateral vagotomy was followed by a very large increase in plasma ANP to 703 +/- 198 pg/ml. Atrial pressures, however, fell after vagotomy from 2 +/- 2 to 1 +/- 1 mmHg. Therefore, the effect was not due to increased atrial stretch. Right atrial ANP levels were also elevated by vagotomy, but left atrial ANP concentrations did not change with vagotomy or hemorrhage. After hemorrhage in vagotomized rats, plasma ANP decreased to 79 +/- 6 pg/ml. After vagotomy, the ANP concentration in the hypothalamic block did not rise in response to hemorrhage. The results indicate that the vagus nerves provide a tonic inhibition of ANP levels in atria and plasma. The results cannot be explained by atrial distension. The results show independence of brain and plasma ANP and uncover a tonic vagal inhibition of ANP release.

Animals↗

PubMed1989

Modulation of urinary kallikrein and plasma renin activities does not affect established hypertension in the fawn-hooded rat.

Fawn-hooded (FH) rats develop low-renin hypertension which is preceded by a decrease in urinary kallikrein. We examined urinary excretion of active and inactive kallikrein in hypertensive FH male rats and matched animals of the ancestral, normotensive Wistar strain. To determine the effects of modulation of salt intake on the kallikrein profile, rats were given standard rat chow (0.39% NaCl), a low-salt diet (0.02% NaCl), or a high-salt diet (standard chow plus water with 1% NaCl). Control FH rats excreted less active kallikrein (p less than 0.02), had similar amounts of inactive kallikrein, and had a higher inactive/active kallikrein ratio (p less than 0.02) than control Wistar rats. Low salt intake increased active kallikrein 136% (p less than 0.002) and 54% (p less than 0.035) in FH and Wistar rats, respectively, but did not change the level of inactive kallikrein or the inactive/active kallikrein ratio. High salt intake had no effect on kallikrein excretion in either strain. Low salt intake did not change blood pressure in either strain in spite of significant changes in plasma renin activity, angiotensin II and active kallikrein excretion. The low urinary active kallikrein and the high inactive/active kallikrein ratio in FH rats do not appear to play a role in the established hypertension in the FH rat, since modulation of these parameters did not cause a significant change in the elevated blood pressure.

Angiotensin II↗

PubMed1989

Plasma atrial natriuretic polypeptide and angiotensin II in rats during anesthesia and volume loading.

These experiments investigated the effect of halothane or fentanyl anesthesia on plasma atrial natriuretic polypeptide (ANP) and angiotensin II (AII) concentrations and the response to a 30% blood volume load in rats. Halothane (1.1% and 2.2% inspired) or fentanyl (50 and 200 micrograms/kg) anesthesia alone produced little change in basal plasma ANP levels but did increase plasma AII levels above unanesthetized baseline concentrations. A 30% blood volume load in conscious rats produced a transient increase in blood pressure and central venous pressure, a threefold rise in plasma ANP, and a decrease in AII. These effects were not significantly altered by fentanyl anesthesia. In contrast, during 2.2% halothane anesthesia the increase in plasma ANP produced by volume loading was greater and the decrease in AII was abolished. These results indicate that fentanyl does not increase basal ANP levels or markedly change the hormonal response to a volume load. Altered responses seen during deep halothane anesthesia may result from cardiovascular changes or a direct stimulation of physiological mechanisms that release ANP.

Angiotensin II↗

PubMed1988

Human intestinal brush border angiotensin-converting enzyme activity and its inhibition by antihypertensive Ramipril.

Angiotensin-converting enzyme (ACE) has been identified as a prominent brush border membrane-bound enzyme of human jejunum. In this study, we purified brush border membrane vesicles enriched in ACE, and characterized the ACE with regard to (a) its stability in the membrane, (b) substrate hydrolysis kinetics compared with pulmonary endothelial ACE, and (c) pharmacologic interaction with Ramipril. These investigations resulted in the following findings. The uninhibited enzyme is stable in native membranes in vitro, with a half-life of 195 +/- 7 h. Kinetic analysis of ACE hydrolysis activity revealed the presence of a single enzyme species, which yielded a high Vmax and displayed a Km similar to purified ACE from lung endothelium. Brush border ACE was inhibited by Ramipril, one of the most specific and potent orally administered ACE inhibitors indicated for hypertension. We determined the brush border ACE value of IC50 = 3 X 10(-9) M Ramipril-diacid, which is the same value for serum and lung ACE. Brush border ACE remains 100% inhibited by 10 microM Ramipril during at least 8 days in vitro. The data indicate that ACE is a prominent jejunal brush border enzyme that behaves pharmacologically and kinetically like its peripheral circulation counterpart. This study suggests that high doses of orally administered ACE inhibitors may affect intestinal epithelial function.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1988

Ramipril inhibition of rabbit (Oryctolagus cuniculus) small intestinal brush border membrane angiotensin converting enzyme.

1. Rabbit small intestinal brush border membranes possessed prominent angiotensin converting enzyme (ACE) activity. 2. Intestinal ACE was located on the lumen surface, as verified by ACE co-enrichment with brush border membrane marker enzymes. 3. Hydrolysis kinetics of rabbit intestinal ACE were comparable to the lung, utilizing the substrate (N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine; the Vmax = 543 +/- 51 mumol/min/g and Km = 0.62 +/- 0.09 mmol/l. 4. Intestinal brush border ACE activity was strongly inhibited by the antihypertensive drug Ramipril, which yielded an IC50 value of 5 nmol/l; the ACE activity remained completely inhibited during 15 days after a single dose of 10 mumol/l Ramipril.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1988

Evidence for extracellular deamination of adenosine in the rat heart.

1. In rat heart perfused with adenosine (10(-6) M), dilazep (10(-4) M) inhibited incorporation of adenosine into nucleotides (an index of nucleoside transport and phosphorylation) to a greater extent (70%) than metabolism to inosine and uric acid (40%) and actually increased the recovery of inosine to 30% of the adenosine infused. 2. Extrapolating for complete inhibition of transport suggested that 60% of adenosine metabolism was intracellular and 40% extracellular. 3. Static incubations of atria also gave an estimate for extracellular metabolism of 40%. 4. Adenosine deaminase was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries. 5. Extracellular deamination may explain the lack of effect of nucleoside transport inhibitors on responses to adenosine in rat heart.

Adenosine↗

PubMed1988

Brain angiotensin in the developing spontaneously hypertensive rat.

There are several factors in the manifestation of high blood pressure in spontaneously hypertensive rats (SHR) which implicate a central role for brain angiotensin II (Ang II). We have measured levels of angiotensin in the brain of SHR and rats of the Wistar-Kyoto strain (WKY). The experiments were carried out in 2-, 4-, 14- and 20-week-old rats. Areas of brain from rats were homogenized and purified with SepPak C-18 cartridges. The levels were measured by radio-immunoassay whose detection limit was 1.95 pg/tube. Significant differences were found between the different age groups and between SHR and controls. In the hypothalamus, there was a consistent elevation of brain Ang II in SHR as compared to WKY in all age groups. Cerebellum also had higher levels in SHR, especially in rats at 2 and 4 weeks of age. Brainstem levels were significantly higher in SHR only in the 14-week-old age group. Plasma levels during these time periods did not differ significantly between the strains. The results demonstrate changes in brain Ang II with development. At an early age, there are high levels of Ang II in the hypothalamus and cerebellum which do not correlate with hypertension but may be important for the development of hypertension. The higher levels of brain Ang II in SHR support the hypothesis that hypertension in SHR is related to brain Ang II activity.

Angiotensin II↗

PubMed1988

Biosynthesis of angiotensinogen and angiotensins by brain cells in primary culture.

This study focuses on the ability of primary rat brain cells in culture to synthesize angiotensinogen, angiotensin I, and angiotensin II. HPLC in combination with radioimmunoassay was used to characterize these compounds. Following incubation with 3H-labeled isoleucine, radioactively labeled angiotensinogen with an approximate molecular weight of 25,000 was identified in both glial and neuronal cells. Other molecular weight forms of angiotensinogen with molecular weights of about 300 and 160,000 were present in both cell types. In addition to angiotensinogen, radioactively labeled angiotensin I and angiotensin II were also synthesized by neuronal and glial cells. These results suggest that glial and neuronal cells can synthesize angiotensinogen, angiotensin I, and angiotensin II in a similar manner shown for the peripheral renin angiotensin system.

Angiotensin I↗

PubMed1988

Renal tubular vasopressin receptors downregulated by dehydration.

Receptors for arginine vasopressin (AVP) were characterized in tubular epithelial basolateral membranes (BL membranes) prepared from the kidneys of male Sprague-Dawley rats. Association of [3H]AVP was rapid, reversible, and specific. Saturation studies revealed a single class of saturable binding sites with a maximal binding (Bmax) of 184 +/- 15 fmol/mg protein and a KD of 0.61 +/- 0.04 nM. IC50S for AVP, lysine vasopressin, and oxytocin were 0.74 nM, 9.7 nM, and greater than 1 microM, respectively. The V2 receptor antagonist was more than 3,700 times as effective in displacing [3H]AVP than was the V1 antagonist. To investigate the physiological regulation of vasopressin receptors, the effects of elevated levels of circulating AVP on receptor characteristics were studied. Seventy-two-hour water deprivation significantly elevated plasma osmolality and caused an 11.5-fold increase in plasma [AVP]. Scatchard analysis revealed a 38% decrease in the number of AVP receptors on the BL membranes from dehydrated animals. The high-affinity binding sites on the BL membranes fit the pharmacological profile for adenylate cyclase-linked vasopressin receptors (V2), which mediate the antidiuretic action of the hormone. We conclude that physiologically elevated levels of AVP can downregulate vasopressin receptors in the kidney.

Animals↗

PubMed1988

Vasopressin stimulates phosphoinositide hydrolysis in LLC-PK1 cells.

LLC-PK1 cells have been shown to possess vasopressin (VP) receptors (V2 type) that are coupled to adenyl cyclase to generate adenosine 3,5'-cyclic monophosphate (cAMP). To determine whether VP also stimulates phosphoinositide (PI) hydrolysis to generate inositol phosphate (IP) and diacylglycerol (DAG) messenger system in LLC-PK1 cells, we measured the release of IP in LLC-PK1 cells in the absence and presence of various concentrations of VP. In addition, we also determined the effect of an increase in osmolality of the incubation medium on VP-stimulated PI hydrolysis in LLC-PK1 cells. The methods involved the incubation of LLC-PK1 cells with [3H]inositol for its incorporation into membrane PI and the measurement of the release of [3H]IP in the presence of LiCl which prevents dephosphorylation. The osmolality of the incubation media was increased from 300 to 600, 900, and 1,200 mosmol/kgH2O by the addition of NaCl and urea. In an isosmotic incubation medium, VP (10(-8) M) produced a 100% increase in PI hydrolysis in LLC-PK1 cells. The effect was much greater at higher concentrations of the hormone. There was no effect of osmolality in VP-stimulated PI hydrolysis in LLC-PK1 cells up to 600 mosmol/kgH2O, but PI hydrolysis decreased significantly when the osmolality of the incubation medium was increased to 900 or 1,200 mosmol/kgH2O. Our results suggest that in LLC-PK1 cells, VP stimulates PI hydrolysis probably through VP receptors that are coupled to phospholipase C. Furthermore, VP-stimulated PI messenger system in LLC-PK1 cells is influenced by osmolality of the extracellular fluid.

Animals↗