Endothelin stimulates sis-inducing factor-like DNA binding activity in CHO-K1 cells expressing ETA receptors.

ET-1, a member of the family of peptides known as endothelins, binds to a G-protein-coupled receptor, ET(A), and stimulates a variety of cellular responses, including contraction, growth, and mitogenesis. ET-1 stimulation of a chinese hamster ovary cell line stably transfected with the ET(A) receptor (CHO/ET(A)) induced formation of SIF (sis-inducing factor), a key component of the STAT (Signal Transducers and Activators of Transcription) pathway, in a concentration-dependent manner. SIF induction was blocked by a specific inhibitor of ET(A), BQ610, and by genistein, a tyrosine kinase inhibitor. This report demonstrates that ET-1 stimulates the STAT pathway of signal transduction through a G-protein-coupled receptor, ET(A), in this stably transfected cell line.

Angiotensin II-induced protein tyrosine phosphorylation in neonatal rat cardiac fibroblasts.

Angiotensin II has been demonstrated to act as a growth factor in rat cardiac fibroblasts. However, the signaling events that lead to fibroblast cell growth in response to angiotensin II remain to be elucidated. This study was designed to determine whether angiotensin II stimulated tyrosine phosphorylation of proteins in cardiac fibroblasts. Immunoblot analysis demonstrated rapid tyrosine phosphorylation of distinct substrates of 125, 95, 46-60, and 44 kDa in response to 10 nM angiotensin II. Tyrosine phosphorylation was maximal at 5 min and persisted for at least 180 min. Additional tyrosine-phosphorylated proteins of 185, 145, and 85 kDa were detected in response to 10 ng/ml platelet-derived growth factor BB. A cluster of 75-80-kDa proteins were phosphorylated in response to angiotensin II, phorbol ester, and platelet-derived growth factor. Angiotensin II-induced tyrosine phosphorylation was unaffected by phorbol ester-sensitive protein kinase C down-regulation and could be partially blocked by pertussis toxin pretreatment. Angiotensin II stimulation resulted in increased cytosolic tyrosine kinase activity which was recovered by immunoprecipitation. Immunoblot analysis demonstrated tyrosine phosphorylation of p44MAPK, and, in addition, we demonstrated for the first time tyrosine phosphorylation of p125FAK, p46SHC, and p56SHC in response to angiotensin II. The finding that angiotensin II and platelet-derived growth factor stimulated tyrosine phosphorylation of p46SHC and p56SHC suggested that this protein may serve as a common tyrosine kinase substrate in the mitogenic signaling cascade induced by G-protein-coupled receptors and growth factors and is consistent with the hypothesis that angiotensin II-induced tyrosine phosphorylation is involved in mitogenic signaling pathways in neonatal rat cardiac fibroblasts.

Angiotensin converting enzyme inhibition in Dahl salt-sensitive rats.

Angiotensin II has previously been reported to have in vivo and in vitro cardiac hypertrophic effects. We used the salt-sensitive Dahl rat genetic strain to separate mechanical (pressure overload) vs. hormonal (renin-angiotensin system) input in cardiac hypertrophy. Blood pressure was significantly increased and left ventricular hypertrophy, as indexed by LV/BW ratios, was present at 7 and 15 days in rats receiving 4% and 8% NaCl compared to the 1% controls. There was no effect of the angiotensin converting enzyme inhibitor, enalapril maleate, on lowering the blood pressure in 8% NaCl-treated animals, however, there was a significant reduction in LV/BW ratio in 8% NaCl-treated animals that received this drug. Left ventricular angiotensinogen mRNA activity was significantly reduced in rats receiving 4% and 8% NaCl. In this model of hypertension the cardiac hypertrophy which develops is largely dependent on mechanical forces though there remains a significant contribution to this process from either circulating or localized angiotensin II production. Regulation of angiotensinogen gene expression in the hypertrophied left ventricle suggests that volume and electrolyte control of angiotensinogen gene expression in the heart and/or hereditary factors are predominant in the control of regulation of this gene in the left ventricle of Dahl rats.

Phosphatidylinositol 4,5-Bisphosphate Phospholipase C and Phosphomonoesterase in Dunaliella salina Membranes.

In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [(3)H]phosphatidylinositol 4,5-bisphosphate (PIP(2)). Based on release of [(3)H]inositol phosphates, the plasma membrane exhibited a PIP(2)-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast ;bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [(3)H]inositol phosphates released were recovered as [(3)H]inositol trisphosphate (IP(3)). These results suggest a plausible mechanism for the rapid breakdown of PIP(2) and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [(3)H]inositol phospholipids during these assays revealed that some of the [(3)H]-PIP(2) was converted to [(3)H]phosphatidylinositol 4-monophosphate (PIP) and to [(3)H]phosphatidyl-inositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP(2)/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free [Ca(2+)]. We also report here that 100 micromolar GTPgammaS stimulates phosphospholipase C activity over a range of free [Ca(2+)]. Together, these results provide evidence that the plasma membrane PIP(2)-phospholipase C of D. salina may be subject to Ca(2+) and G-protein regulation.

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity.

We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.

Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock.

The inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) comprise 14.8, 1.2, and 0.3 mol %, respectively, of Dunaliella salina phospholipids. In isolated plasma membrane fractions, PIP and PIP2 are highly concentrated, together comprising 9.5 mol % of plasmalemma phospholipids. The metabolism of these inositol phospholipids and phosphatidic acid (PA) is very rapid under normal growth conditions. Within 5 min after introduction of 32Pi into the growth medium, over 75% of lipid-bound label was found in these quantitatively minor phospholipids. Within 2 min after a sudden hypoosmotic shock, the levels of PIP2 and PIP dropped to 65 and 79%, respectively, of controls. Within the same time frame, PA rose to 141% of control values. These data suggest that a rapid breakdown of the polyphosphoinositides may mediate the profound morphological and physiological changes which allow this organism to survive drastic hypoosmotic stress. In contrast to hypoosmotic shock, hyperosmotic shock induced a rise in PIP2 levels to 131% of control values, whereas the level of PA dropped to 56% of controls after 4 min. These two different types of osmotic stress affect inositol phospholipid metabolism in a fundamentally opposite manner, with only hypoosmotic shock inducing a net decrease in polyphosphoinositides.

A Comparison of the Effects of Chilling on Thylakoid Electron Transfer in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.).

Experiments comparing the photosynthetic responses of a chilling-resistant species (Pisum sativum L. cv Alaska) and a chilling-sensitive species (Cucumis sativus L. cv Ashley) have shown that cucumber photosynthesis is adversely affected by chilling temperatures in the light, while pea photosynthesis is not inhibited by chilling in the light. To further investigate the site of the differential response of these two species to chilling stress, thylakoid membranes were isolated under various conditions and rates of photosynthetic electron transfer were determined. Preliminary experiments revealed that the integrity of cucumber thylakoids from 25 degrees C-grown plants was affected by the isolation temperature; cucumber thylakoids isolated at 5 degrees C in 400 millimolar NaCl were uncoupled, while thylakoids isolated at room temperature in 400 millimolar NaCl were coupled, as determined by addition of gramicidin. The concentration of NaCl in the homogenization buffer was found to be a critical factor in the uncoupling of cucumber thylakoids at 5 degrees C. In contrast, pea thylakoid membranes were not influenced by isolation temperatures or NaCl concentrations. In a second set of experiments, thylakoid membranes were isolated from pea and cucumber plants at successive intervals during a whole-plant light period chilling stress (5 degrees C). During wholeplant chilling, thylakoids isolated from cucumber plants chilled in the light were uncoupled even when the membranes were isolated at warm temperatures. Pea thylakoids were not uncoupled by the whole-plant chilling treatment. The difference in integrity of thylakoid membrane coupling following chilling in the light demonstrates a fundamental difference in photosynthetic function between these two species that may have some bearing on why pea is a chilling-resistant plant and cucumber is a chilling-sensitive plant.

A Comparison of the Effects of Chilling on Leaf Gas Exchange in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.).

The effects of chilling on the photosynthesis of a chilling-resistant species, pea (Pisum sativum L. cv Alaska) and a chilling-sensitive species, cucumber (Cucumis sativus L. cv Ashley) were compared in order to determine the differences in the photosynthetic chilling sensitivity of these two species. For these experiments, plants were chilled (5 degrees C) for different lengths of time in the dark or light. Following a 1 hour recovery period at 25 degrees C, photosynthetic activity was measured by gas exchange (CO(2) uptake and H(2)O release), quantum yield, and induced chlorophyll fluorescence. The results show that pea photosynthesis was largely unaffected by two consecutive nights of chilling in the dark, or by chilling during a complete light and dark cycle (15 hours/9 hours). Cucumber gas exchange was reduced by one night of chilling, but its quantum yield and variable fluorescence were unaffected by dark chilling. However, chilling cucumber in the light led to reduced CO(2) fixation, increased internal leaf CO(2) concentration, decreased quantum yield, and loss of variable fluorescence. These results indicate that chilling temperatures in conjunction with light damaged the light reactions of photosynthesis, while chilling in the dark did not.

The Influence of Dark Adaptation Temperature on the Reappearance of Variable Fluorescence following Illumination.

The effect of chilling temperatures (5 degrees C) on chlorophyll fluorescence transients was used to study chilling-induced inhibition of photosynthesis in plant species with differing chilling sensitivities. A Brancker SF-20 fluorometer was used to measure induced fluorescence transients from both attached and detached leaves of chilling-sensitive cucumber (Cucumis sativus L. cv Ashley) and chilling-resistant pea (Pisum sativum L. cv Alaska). The rate of reappearance of the variable component of fluorescence (F(v)), following a period of illumination at 25 degrees C, was dependent on the temperature at which the leaf was allowed to dark adapt in chilling-sensitive cucumber, but not in chilling-resistant pea. In cucumber, dark adaptation at 25 degrees C following illumination resulted in a much faster return of F(v) than dark adaptation at 5 degrees C following illumination. However, F(v) reappearance during the dark adaptation period in chilling-resistant pea was temperature independent. The difference in the temperature response of F(v) following illumination correlated with temperature sensitivity of these two species. The process responsible for the difference in F(v) may represent a site of chilling sensitivity in the photosynthetic apparatus.

Chloroplast ultrastructure, chlorophyll fluorescence, and pigment composition in chilling-stressed soybeans.

Shoots of 16-day-old soybeans (Glycine max L. Merr. cv Ransom) were chilled to 10 degrees C for 7 days and monitored for visible signs of damage, ultrastructural changes, perturbations in fluorescence of chlorophyll (Chl), and quantitative changes in Chl a and b and associated pigments. Precautions were taken to prevent the confounding effects of water stress. A technique for the separation of lutein and zeaxanthin was developed utilizing a step gradient with the high performance liquid chromatograph. Visible losses in Chl were detectable within the first day of chilling, and regreening did not occur until the shoots were returned to 25 degrees C. Ultrastructurally, unstacking of chloroplast grana occurred, and the envelope membranes developed protrusions. Furthermore, the lipids were altered to the point that the membranes were poorly stabilized by a glutaraldehyde/osmium double-fixation procedure. Chl fluorescence rates were greatly reduced within 2 hours after chilling began and returned to normal only after rewarming. The rapid loss of Chl that occurred during chilling was accompanied by the appearance of zeaxanthin and a decline in violaxanthin. Apparently a zeaxanthin-violaxanthin epoxidation/de-epoxidation cycle was operating. When only the roots were chilled, no substantial changes were detected in ultrastructure, fluorescence rates, or pigment levels.