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Biomedical subjects

A Menachery

Publications and source records attributed to A Menachery.

13 recordsLinked to original sources

Controlling cell destruction using dielectrophoretic forces.

Measurements are reported of the main factors, namely the AC voltage frequency and magnitude, that were observed to influence the number of cells destroyed during dielectrophoresis (DEP) experiments on Jurkat T cells and HL60 leukemia cells. Microelectrodes of interdigitated and quadrupolar geometries were used. A field-frequency window has been identified that should be either avoided or utilised, depending on whether or not cell damage is to be minimised or is a desired objective. The width and location of this frequency window depends on the cell type, as defined by cell size, morphology and dielectric properties, and is bounded by two characteristic frequencies. These frequencies are the DEP cross-over frequency, where a cell makes the transition from negative to positive DEP, and a frequency determined by the time constant that controls the frequency dependence of the field induced across the cell membrane. When operating in this frequency window, and for the microelectrode designs used in this work, cell destruction can be minimised by ensuring that cells are not directed by positive DEP to electrode edges where fields exceeding 30-40 kV/m are generated. Alternatively, this field-frequency window can be exploited to selectively destroy specific cell types in a cell mixture.

Cell Fractionation↗

Comparison of protein phosphorylation patterns produced in adrenal cells by activation of cAMP-dependent protein kinase and Ca-dependent protein kinase.

Bovine adrenal fasciculata cells, exposed to either ACTH or AII, synthesize glucocorticoids at an enhanced rate. It is generally accepted that the signaling pathways triggered by these two peptides are not identical. ACTH presumably acts via a cAMP-dependent protein kinase (PKA) and AII, via a calcium-dependent protein kinase. We have found that either peptide hormone stimulates synthesis of a mitochondrial phosphoprotein pp37, leading to accumulation of its proteolytically processed products pp30 and pp29. On the basis of a number of criteria, this 37 kDa protein is the bovine homolog of the 37 kDa protein that we have characterized in rodent steroidogenic tissue (Epstein L. F. and Orme-Johnson N. R.: J. Biol. Chem 266 (1991) 19,739-19,745). Further, bovine pp37 is phosphorylated when PKA or protein kinase C (PKC) is activated directly by (Bu)2 cAMP or PMA, respectively. These studies indicate that either pp37 is a common substrate for PKA and PKC in these cells or there is a common downstream kinase, which is activated by exposure to either ACTH or AII. Rat adrenal glomerulosa cells, exposed to either ACTH or AII, show an enhanced rate of mineralocorticoid synthesis. As for bovine fasciculata cells, it is thought that the signaling pathway triggered by ACTH differs from that triggered by AII. As we found for bovine fasciculata, pp37 is phosphorylated when the rat cells are exposed to either peptide hormone. However, in contrast to the finding for bovine fasciculata, while exposure of the rat glomerulosa cells to (Bu)2cAMP does cause the synthesis of pp37, exposure of the cells to PMA does not. Taken together, these findings provide further evidence that the subcellular signaling events, triggered by the action of AII on bovine adrenal fasciculata and rat adrenal glomerulosa cells, differ. Further, the fact, that pp37 is phosphorylated only when the rate of steroidogenesis is enhanced, reaffirms its potential involvement in the signaling pathway that causes stimulation of steroid hormone biosynthesis.

Adrenal Glands↗

Red cell sodium-proton exchange is increased in Dahl salt-sensitive hypertensive rats.

To investigate the relationship between red blood cell Na+/H+ exchange (EXC) and genetic factors in hypertension, we studied the maximal rate of the antiporter (mmol/liter cell x hr; flux units = FU) in three strains of genetically hypertensive rats. Salt-resistant Dahl rats (DR) were normotensive under low (0.02%) and high (8%) NaCl diets, while salt-sensitive Dahl rats (DS) became markedly hypertensive after four weeks on the high-NaCl diet. Na+/H+ exchange did not differ between DR and DS rats when both were fed with the low-NaCl diet (mean +/- SE, 31 +/- 3, N = 15, vs. 29 +/- 3 FU, N = 14). On the high-NaCl diet, the DR strain did not exhibit significant changes in blood pressure and antiporter activity, but the DS rats significantly increased their blood pressure and Na+/H+ exchange (57 +/- 4 FU, N = 13) versus DR rats (38 +/- 3 FU, N = 15, P < 0.02). DS rats also significantly increased blood pressure and antiporter activity when fed with high-NaCl diet for one week. These data indicate that high NaCl intake per se does not increase Na+/H+ EXC because the control DR strain did not exhibit transport and blood pressure alterations as observed in the DS strain. Milan hypertensive and spontaneously hypertensive rats (Charles River substrain) had higher blood pressures than Milan and Wistar-Kyoto normotensive rats when they were maintained for four weeks on a 1.5% NaCl diet; however, no differences were seen among normotensive and hypertensive strains in Na+/H+ exchange activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Dissociation in plasma renin and adrenal ANG II and aldosterone responses to sodium restriction in rats.

In rats, plasma renin activity (PRA) increases sharply, reaching a plateau within hours of sodium restriction. Plasma aldosterone increases gradually, not reaching a plateau for 1-2 days. To determine whether this dissociation is secondary to the time needed to modify adrenal sensitivity to angiotensin II (ANG II) and to assess the role of locally produced ANG II in this process, rats were salt restricted for 0-120 h. Plasma hormone levels were assessed, adrenal ANG II was measured, and basal and ANG II (1 x 10(-8) M)-stimulated steroidogenesis were determined in vitro. Although PRA attained an elevated plateau within 8 h, plasma aldosterone did not peak until after 48 h of sodium depletion. The in vitro aldosterone sensitivity to exogenous ANG II was not apparent until rats had been salt restricted for 16 h. A plateau (4-fold increase above the ANG II response on high salt) was achieved between 24 and 48 h. Adrenal ANG II also exhibited a similar delayed response that correlates significantly with changes in aldosterone biosynthesis and late pathway activity. Thus the dissociation between PRA and plasma aldosterone may be secondary to a lag in the zona glomerulosa's (ZG) steroidogenic response to ANG II as well as a parallel lag in tissue ANG II production, suggesting that changes in tissue ANG II may mediate ZG sensitivity to ANG II during sodium deprivation.

Aldosterone↗

The effect of sodium intake on angiotensin content of the rat adrenal gland.

To determine whether dietary sodium intake modifies the generation of adrenal-produced angiotensins and/or their relative proportions, Sprague-Dawley rats were maintained on a low (0.02%), normal (0.4%), or high (1.5%) sodium intake for 5 days. The animals were then killed by decapitation at 0900 h, and their adrenal glands were removed and dissected into two parts: capsular tissue, containing the zona glomerulosa (ZG), and the decapsulated adrenal gland. The tissue was frozen in liquid nitrogen and extracted, and the individual angiotensins [angiotensin-II (AII), angiotensin-III (AIII), angiotensin-I (AI), and Des-Asp-angiotensin-I (Des-Asp-AI)] were separated by HPLC and quantitated by RIA. On a normal sodium intake, the molar contents of the four angiotensins were similar in ZG, ranging from 3.1-6.6 pmol/g, although AII was present in a 60-70% higher concentration than AIII. In the decapsulated adrenal, the concentrations of the various angiotensins were again similar, but the absolute levels (per g tissue) were significantly (P less than 0.02) less than those in the ZG layer. With sodium restriction, the AII content increased more than 2-fold in the ZG, but not in the decapsulated adrenal tissue. In contrast, both AI and Des-Asp-AI significantly (P less than 0.01) decreased with sodium restriction, so that their contents on the low salt diet were only 15-20% of those observed on the high sodium diet. Thus, there was an inverse correlation (P less than 0.001) between the salt content of rat chow and the AII content of the ZG. The correlation between salt intake and AI as well as Des-Asp-AI levels was direct and significant (P less than 0.02). The AIII level in the ZG was similar on all diets. After a lag period, ZG AII increased sharply between 16-48 h of sodium restriction. These data document that sodium intake has a profound effect on the angiotensin content of the ZG, with sodium restriction substantially increasing the levels of AII while reducing the level of its substrate, AI. This also appears to be unique for glomerulosa cells, as in the decapsulated adrenal gland there is little if any change with sodium restriction. We conclude that these sodium-mediated changes in tissue AII production may be involved in the increased responsiveness of glomerulosa cells to aldosterone secretagogues during sodium restriction.

Adrenal Glands↗

Potassium-stimulated angiotensin release from superfused adrenal capsules and enzymatically dispersed cells of the zona glomerulosa.

The cells of the adrenal cortex contain angiotensin-II (AII), but whether this peptide is synthesized there (vs. internalized from the systemic circulation), whether it is secreted, and whether it is important in aldosterone production remain uncertain. To address these issues, we studied AI and AII release from superfused rat adrenal capsules and dispersed glomerulosa cells. Superfused adrenal capsules released 7-fold more AII in 270 min than the capsules originally contained (495 +/- 101 fmol AII/rat released vs. 66 +/- 8 fmol AII/rat tissue content). The amount of AI released in the same period only slightly exceeded the tissue content. In response to higher potassium concentrations in the medium (9 vs. 3.6 mM K+), adrenal capsules and dispersed glomerulosa cells both released significantly more AI and AII into the superfusate. This release of AI and AII was oscillatory. The oscillations occurred in each of 15 experiments, with a period of 45-90 min. Decapsulated adrenal glands (the zona faciculata/reticularis plus medulla) also contained and released AII, but did not respond to potassium stimulation. There was a highly significant correlation between AII and aldosterone release. This was especially apparent if aldosterone secretion was examined during oscillations of AII release (r = 0.97; P less than 0.0001). We conclude that AII is synthesized in the zona glomerulosa and can be released in response to stimuli. The close correlation between AII and aldosterone secretion suggests that locally produced AII may play an important role in aldosterone biosynthesis.

Adrenal Glands↗

Rate of activation of renin-angiotensin-aldosterone axis and sodium intake in rats.

When sodium intake in the rat is reduced abruptly from the typical high level to a very low level (0.02%), sodium excretion falls exponentially, with a half time of 2-3 h. The result is that the rat achieves external sodium balance, in which intake equals excretion, on the new low intake within a few hours. In this study, we assessed the rate of activation of the renin-angiotensin-aldosterone axis and its contribution to blood pressure during that interval. Plasma renin activity and angiotensin II concentration had risen sharply within 8 h and did not change over the next 40 h. Plasma aldosterone concentration, on the other hand, continued to rise over 48 h. Within 8 h, blood pressure dependency on angiotensin II had increased sharply, as assessed by depressor responses to an angiotensin antagonist (Sar1-Ala8-angiotensin II) and to converting-enzyme inhibition (captopril). The depressor response to neither agent changed over the next 40 h. The pressor response to angiotensin II was blunted significantly by 8 h and also did not change over the next 40 h. The findings indicate that the rapid tempo of sodium homeostasis in the rat is matched by an equally rapid tempo of activation of the renin-angiotensin system, although the factors responsible for aldosterone release are probably more complex. Experiments to assess the renin-angiotensin system in the rat must be designed with this rapid tempo in mind.

Aldosterone↗

Sodium-mediated modulation of aldosterone secretion: impact of converting enzyme inhibition on rat glomerulosa cell response to angiotensin-II.

Sodium restriction enhances the aldosterone response to angiotensin-II (AII) in normal rats, but not in spontaneously hypertensive rats (SHR). To determine whether a change and/or abnormality in the circulating or adrenal renin-angiotensin systems are responsible for these observations, three groups of animals were studied on a low sodium diet with and without the administration of a converting enzyme inhibitor (enalapril). Sprague-Dawley and Wistar-Kyoto (normotensive rat strains) and SHR were placed on low sodium (0.1%) for 9 days, the last 4 days of which enalapril was administered to half of the animals. In all groups enalapril treatment resulted in a significant (P less than 0.001) reduction in blood pressure, an increase in renin activity, and a reduction in plasma aldosterone when all of the animals were considered together, although the change in blood pressure achieved statistical significance only in the Wistar-Kyoto rats. Additionally, basal aldosterone output from isolated glomerulosa cells was lower in the normotensive animals pretreated with enalapril. However, despite the evidence for inhibition of converting enzyme, there was no change in the hypertensive animals. Thus, neither locally nor systemically generated AII appear to participate in the maintenance of the increased aldosterone responsiveness to AII with sodium restriction. Furthermore, they do not appear to contribute to the altered adrenal responsiveness to AII with sodium restriction in SHR. These data provide further support for the hypothesis that as yet undefined glomerulosa intracellular mechanisms are altered by dietary sodium restriction in normotensive, but not hypertensive, rats.

Adrenal Medulla↗

Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat.

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N6 O2'-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3.5 mmol/l), significantly (P less than 0.01) increased aldosterone output by at least 1.5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P less than 0.01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 +/- 14 vs 105 +/- 19 pmol/10(6) cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion. Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P less than 0.01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine. These observations suggest that: the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.

Adrenal Cortex↗

Calcium, a "third messenger" of cAMP-stimulated adrenal steroid secretion.

This study examines the role of extracellular calcium and calcium mobilization from intracellular stores in mediating cAMP-stimulated steroid secretion by rat adrenal glomerulosa cells (GC) and fasciculata cells (FC). When GC were incubated acutely in a calcium-deficient buffer, cAMP failed to significantly increase aldosterone secretion above base line. Aldosterone secretion, however, rose from 17 +/- 2 to 32 +/- 4 ng/10(6) cells (P less than 0.01) as calcium in the medium was increased from 0 to 3.5 mM. In contrast, cAMP-stimulated corticosterone production by FC was not influenced by changes in the external calcium concentration. Lanthanum (10(-4) M), an inhibitor of calcium influx, reduced cAMP-stimulated aldosterone secretion from 69 +/- 10 to 42 +/- 5 ng/10(6) cells (P less than 0.01) but failed to alter cAMP-stimulated fasciculata steroidogenesis. Depletion of intracellular calcium stores, achieved by incubating with EGTA, markedly blunted cAMP-stimulated corticosterone secretion in GC from 666 +/- 126 to 32 +/- 6 ng/10(6) cells (P less than 0.01), and cAMP-stimulated corticosterone secretion in FC from 2,223 +/- 407 to 414 +/- 58 ng/10(6) cells (P less than 0.01). TMB-8, a putative inhibitor of intracellular calcium mobilization, markedly inhibited (P less than 0.01) cAMP-stimulated aldosterone secretion by GC from 469 +/- 31 to 48 +/- 8 ng/10(6) cells and corticosterone secretion by FC from 9,867 +/- 1,821 to 2,832 +/- 586 ng/10(6) cells. These observations suggest that cAMP activation of adrenal steroidogenesis requires the release of calcium from intracellular stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗

The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells.

We evaluated changes in cytosolic calcium concentration (Ca++) and steroidogenesis in rat adrenal glomerulosa cells (GC) stimulated with potassium (K+) or angiotensin II (AII). Cytosolic Ca++ concentration was determined using the Ca++-sensitive, fluorescent dye QUIN 2. Raising extracellular K+ increased cytosolic Ca++ from 267 +/- 23 nM at 3.7 mM K+ to a maximum of 377 +/- 40 nM at 8.7 mM K+ (p less than 0.01, N = 23). AII also increased cytosolic Ca++ from 238 +/- 20 nM to a maximum of 427 +/- 42 nM at 10(-7) M (p less than 0.01, N = 16). In parallel studies, K+ and AII stimulated aldosterone secretion from QUIN 2-loaded GC at concentrations similar to those which raised cytosolic Ca++. QUIN 2-loaded cells were as responsive steroidogenically as unloaded cells and showed trypan blue exclusion of 98% suggesting that QUIN 2 did not compromise cellular viability. These results provide direct support for a role of cytosolic Ca++ as a second messenger during stimulation of aldosterone secretion by both K+ and AII.

Adrenal Cortex↗

Decreased adrenal responsiveness to angiotensin II: a defect present in spontaneously hypertensive rats. A possible model of human essential hypertension.

30% of patients with essential hypertension have a decreased adrenal response to angiotensin II (A II) on a low but not a high sodium intake. They also have a compensatory increase in the activity of the renin-angiotensin system best documented in a sodium-restricted state.To assess whether such a mechanism could account for the hypertension in genetically hypertensive rats, adrenal responsiveness to A II was determined in three groups of rats; spontaneously hypertensive rats (SHR), normotensive Wistar rats (WKY), and normotensive Sprague-Dawley rats (SDR). Animals in each group were placed on either a low or high sodium diet for 14 d with balance assessed by sodium excretion. The animals were then decapitated, blood was obtained for plasma renin activity (PRA), A II and aldosterone and adrenals isolated for the preparation of purified glomerulosa cells. The cells were incubated in Krebs-Ringer bicarbonate solution, containing bovine serum albumin, for 60 min in the absence and presence of increasing concentrations of A II. The PRA, basal aldosterone output, and adrenal sensitivity to A II were similar in the three groups of rats on the high sodium diet. On the low sodium diet the SHR had a significantly (P < 0.01) higher PRA (25+/-7 ng/ml per h) than either the WKY (12+/-2 ng/ml per h) or the SDR (7+/-1 ng/ml per h) and lower basal aldosterone output (68+/-17 vs. 154+/-43 and 197+/-21 ng/10(6) cells per h, respectively). In addition, the slope of the A II dose response curve was more shallow (P < 0.01) in the cells from the SHR than those obtained from the WKY and SDR.Thus, the SHR PRA and aldosterone responses to sodium restriction and aldosterone response to A II were similar to that previously described in a subgroup of patients with essential hypertension suggesting that the SHR will serve as a model for exploring the mechanism(s) responsible for the hypertension in these patients.

Adrenal Glands↗

Angiotensin II's role in mediating angiotensin I- and tetradecapeptide-induced steroidogenesis by rat glomerulosa cells.

To evaluate the role of angiotensin II (A II) in mediating the steroidogenic response to angiotensin I (A I) and tetradecapeptide, rat glomerulosa cells were incubated with each peptide in the presence or absence of an angiotensin-converting enzyme inhibitor (captopril or the nonapeptide bradykinin-potentiating factor). Both A I and tetradecapeptide increased aldosterone secretion in a dose-dependent fashion, but were considerably less effective (P less than 0.001) than the same dose (2.4 X 10(-9) M) of A II. In addition, both A I and tetradecapeptide caused a dose-dependent increase in A II accumulation in the incubation media, indicating that part of their steroidogenic effect is indirect via conversion to the octapeptide. While captopril (1.0 X 10(-4) M) almost completely blocked A I (2.4 X 10(-8) M)-induced A II accumulation, it caused only a 50% reduction (P less than 0.01) in aldosterone output. The nonapeptide-converting enzyme inhibitor (2.3 X 10(-6) M) produced a similar blockade. This lack of complete inhibition of A I-induced steroidogenesis suggests that A I also has a direct effect on the glomerulosa cells, i.e. 50% of the activity of A I is due to intrinsic activity. On the other hand, converting enzyme inhibitors did not affect tetradecapeptide-induced aldosterone output or A II accumulation, making it impossible to determine if it has a direct steroidogenic effect. The failure of converting enzyme inhibitors to modify tetradecapeptide-induced accumulation of A II suggests that an enzyme other than converting enzyme is responsible for its generation.

Adrenal Glands↗