Unesterified long chain fatty acids inhibit the binding of single chain urokinase to the urokinase receptor.

The interaction of single chain urokinase with its receptor accelerates plasminogen activator activity on cell surfaces and induces intracellular signalling in several cell types. To date, no physiologic inhibitor of this binding has been identified. We report that the binding of scuPA to its cellular receptor is inhibited by long chain fatty acids such as oleic acid (C18, delta 9) at physiological plasma concentrations. Inhibition of single chain urokinase binding to human trophoblastic cells by long chain fatty acids was dose-dependent and saturable. Fifty percent of the binding was inhibited at an oleic acid concentration of 27 microM, while inhibition was maximal (75%) at 150 microM oleic acid. The inhibitory potency of oleic acid was unaffected by fatty acid free albumin or human plasma. Inhibition of single chain urokinase binding by free fatty acid analogues was critically dependent on chain length (> C14 required for inhibition) and was proportional to the extent of unsaturation. Only the fraction of specific scuPA binding to trophoblasts that was dependent on uPAR was susceptible to inhibition by oleic acid, while binding of scuPA to vitronectin, thombospondin, and the alpha 2-macroglobulin receptor/low-density lipoprotein-related receptor was not. [3H]Oleic acid bound specifically to recombinant soluble uPAR in a 1:1 molar ratio in the presence or absence of plasma and totally blocked its specific binding to a cell line expressing glycosyl phosphatidylinositol-linked single chain urokinase. These results indicate that oleic acid and other unsaturated long chain free fatty acids may serve as physiologic regulators of proteolytic events and intracellular signalling that depend upon the interaction of urokinase with its receptor.

ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger.

Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from 32P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na+] inhibition profile for 45Ca uptake. Thus, ATP depletion inhibits both the Ca2+ influx and Ca2+ efflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Na+ and Ca2+ with the transporter. The latter effect appears to be related to changes in the actin cytoskeleton.

Stable expression of the cardiac sodium-calcium exchanger in CHO cells.

A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.4 cells stably expressed substantial exchange activity and exchanger protein as shown by immunoprecipitation. Exchange activity was quantified as 45Ca2+ influx that depended on both increasing intracellular Na+ and lowering the concentration of external Na+. Replacing external Na+ with K+ slightly increased 45Ca2+ uptake by CK1.4 cells with basal Na+ and greatly increased 45Ca2+ uptake by Na(+)-loaded cells. Neither exchange activity nor exchanger protein was detected in the nontransfected parental line. By contrast to CK1.4 cells, replacing external Na+ with K+ decreased 45Ca2+ uptake in the nontransfected cells whether or not they were Na+ loaded. Changes in cytosolic free Ca2+ determined with fura-2 were consistent with the 45Ca2+ uptake data. Analysis of poly(A)(+)-RNA by Northern blot confirmed that CK1.4 cells, but not the parental line, expressed the exchanger. Expression of the exchanger was also observed in aortic myocytes and a renal epithelial cell line (LLC-MK2) but not in other lines of renal epithelial cells (MDCK, LLC-PK1) or human dermal fibroblasts. The cardiac exchanger produced substantial 45Ca2+ efflux from CK1.4 cells in response to hormone-evoked release of stored Ca2+. CK1.4 cells are an attractive model for studies of the regulation of the cardiac exchanger because they stably express sufficient exchanger for biochemical and immunological analysis.

Cloning and expression of the bovine cardiac sodium-calcium exchanger.

Two clones (p17 and p13), each containing the complete coding sequence for the bovine cardiac Na+/Ca2+ exchanger, were obtained from a lambda gt10 cDNA library by screening with cDNA probes from the canine exchanger. The coding sequence of clone p17 was 92 and 98% identical to the canine cDNA at the nucleotide and amino acid levels, respectively. Nine of the 21 amino acid differences between the two exchangers were found within the 32-amino acid signal sequence. The sequenced portions of the 3' untranslated regions of the cow and dog clones were 88% identical. Na+/Ca2+ exchange activity was expressed in Xenopus laevis oocytes injected with cRNA from clone p17, and in COS cells transfected with expression vectors containing p17. Immunoprecipitation of 35S-labeled proteins from transfected cells with an antibody against the N-terminal portion of the bovine exchanger showed the presence of a 120-kDa protein corresponding to the intact cardiac exchanger. The second bovine clone (p13) did not express exchange activity in either of the above expression systems, presumably because it contained a 300-bp insert with multiple stop codons which interrupted the coding sequence. Comparison of the 5' untranslated regions of p13 and p17 revealed a 156-bp segment in p17 that was apparently spliced out of p13. This segment contained a short open reading frame. A chimera encoding the 5' untranslated region of p13 and the coding sequence of p17 exhibited only a modest (74%) increase in expressed exchange activity in transfected cells compared to p17, suggesting that the presence of the upstream open reading frame in p17 did not greatly reduce translation efficiency. The results suggest that alternate splicing mechanisms may be involved in processing mRNA for the bovine cardiac exchanger.

The effect of lowered extracellular Na+ concentration on ultraviolet light-induced relaxation of vasoconstricted rabbit isolated thoracic aorta.

The effect of lowering extracellular ion concentration on ultraviolet (UV) light-induced photorelaxation of norepinephrine(NE)-constricted rabbit isolated thoracic aorta was investigated. The magnitude of the photorelaxation response (similar to acetylcholine-induced, but not nitroprusside-induced, relaxation) progressively declined, in the absence of an effect on NE-induced vasoconstriction, as the total extracellular ion concentration was progressively reduced. This diminution in the photorelaxation response was duplicated by isosmotic lowering of the extracellular concentration of Na+, but not other ions, from 145 to 25 mM and was not restored by the replenishment of the Na+ deficiency by equimolar amounts of mannitol or Li+. In contrast, choline fully substituted for Na+. These findings suggest a fundamental difference in the ion dependency (and, hence, the mechanisms) of UV-induced photorelaxation and the vasorelaxations induced by acetylcholine or sodium nitroprusside.

Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells.

The octapeptide [Ile5]angiotensin II (ANG II), which is the principal circulating hormone of the renin-angiotensin system, could modulate or mediate cardiac hypertrophy via indirect effects, through increases in total peripheral vascular resistance, or by direct effects on cardiac cells, which result in increased protein synthesis and cell growth. In this study we determined whether ANG II stimulated protein synthesis and cell growth in cultures of embryonic chick myocytes. After 3 h of exposure to ANG II, there were significant increases in total cellular protein at 120, 144, and 168 h and in the relative rate of protein synthesis at 120 and 144 h. There was a significant increase in the fractional rate of protein synthesis of 32.2% (0.0119 +/- 0.0008 h-1 for ANG II stimulated and 0.0090 +/- 0.0003 h-1 for control). The stimulatory effects of ANG II on protein synthesis and cell growth were inhibited by the ANG II antagonist [Sar1,Ile8]ANG II and the hexapeptide ANG II-(3-8). ANG II significantly increased total RNA levels in myocytes, at 12 h after exposure to the peptide. The stimulatory effect of ANG II (32%) on total cellular protein was slightly greater than that seen with norepinephrine (20%) in contrast to the greater stimulatory effect seen with phorbol 12,13-dibutyrate (47%). ANG II and [Sar1,Ile8]ANG II each stimulated increases in cytosolic-free Ca2+, whereas ANG II-(3-8) did not. Growth-related effects of changes in the chronotropic state of the myocytes were excluded, in that, ANG II-stimulated increases in protein synthesis and cellular protein were not inhibited by potassium chloride depolarization of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Renin-angiotensin system involvement in pressure-overload cardiac hypertrophy in rats.

We have recently shown that the octapeptide angiotensin II is a potent stimulus of protein synthesis and growth in cultured cardiomyocytes. The present study was performed to determine if the renin-angiotensin system was involved in regulating cardiac cell growth in vivo. The pressure-overload cardiac hypertrophy model that develops in abdominal aorta-constricted rats was studied. At 7 and 15 days after abdominal aorta constriction, rats developed significant left ventricular hypertrophy. The increase in left ventricular mass was completely prevented in animals fed the angiotensin-converting enzyme inhibitor, enalapril maleate (0.2 mg/ml) in their drinking water. Cardiac afterload was the same in both groups of animals in that carotid artery pressures were not different in conscious awake aortic-constricted animals receiving and not receiving enalapril. These data suggest a direct growth effect of angiotensin II on the left ventricle and indicate a role for the renin-angiotensin system in the cardiac hypertrophy that develops in response to pressure overload. The presence and chamber localization of angiotensinogen mRNA was determined using Northern hybridization and S1 nuclease mapping analysis. Angiotensinogen mRNA, as determined by dot-blot hybridization analysis, was significantly increased in hypertrophied left ventricles at both 7 and 15 days after the surgery, when compared with sham-operated controls. The activity of the circulating renin-angiotensin system, as indexed by plasma renin activity was increased at 1 day following surgery [6.0 +/- 2.0 ng.ml-1.h-1 angiotensin I (control) vs. 41.8 +/- 10.9 ng.ml-1.h-1 angiotensin I (experimental)], but returned to control values by day 3 postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Sar1]angiotensin II receptor-mediated stimulation of protein synthesis in chick heart cells.

Cardiac hypertrophy is a process that occurs in response to various mechanical or hormonal stimuli. Stimulation of the renin-angiotensin system is involved in the process of cardiac hypertrophy through mechanisms related to increased peripheral vascular resistance and increased cardiac afterload. In this study we determined whether [Sar1]angiotensin II (ANG II) directly stimulated protein synthesis and cell growth in embryonic chick myocytes in cell culture. Eighteen-day-old embryonic chick myocytes in subconfluent cell culture, incubated in a chemically defined serum-free media, showed a significant increase in total protein content, 18.5, 26.2, and 22.2%, respectively, when exposed to [Sar1]ANG II (1 microM/day) for 5, 7, and 9 days, respectively. The increase in total protein resulted in part from an increase in the fractional protein synthesis rate of 21.7, 16.5, and 14.9% at 5, 7, and 9 days, respectively. Total DNA and RNA levels did not change significantly following a 4-day exposure to [Sar1]ANG II in subconfluent culture. The relative rate of protein synthesis, determined by pulse labeling for 3 h with [3H]phenylalanine, showed increases of 23.4, 22.9, and 17.8% over control after 4, 5, and 6 days of exposure to [Sar1]ANG II. The incorporation of [3H]phenylalanine was blocked by the specific ANG II-receptor antagonist [Sar1,Ile8]ANG II. The data demonstrate a receptor-mediated increase in the rate of protein synthesis in cultured chick myocytes in response to [Sar1]ANG II, with a resultant increase in total cellular protein. This angiotensin peptide appears to directly stimulate protein synthesis in cultured embryonic chick myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal expression of renin, angiotensinogen, and atriopeptin genes in chick heart.

The renin-angiotensin and atriopeptin systems play important roles in the regulation of volume and fluid homeostasis. The two systems have opposing physiologic actions in a number of tissues. Experiments were performed to determine whether there were differences in the developmental expression of the genes for renin, angiotensinogen, and atriopeptin. Using RNA dot blot analysis, we compared levels of gene activity for renin, angiotensinogen, and atriopeptin in right atria, left atria, right ventricle, and left ventricle, from 18-day in ovum and 10-day old White Leghorn chicks. In 18-day embryonic chick heart there was expression of atriopeptin mRNA predominantly in the left and right ventricles. At this age, atriopeptin message was expressed in all four cardiac chambers, left ventricle greater than right ventricle greater than right atria greater than left atria. Renin and angiotensinogen mRNA was expressed in all cardiac chambers with reduced expression in left atria. In 10-day old chicks, renin, angiotensinogen, and atriopeptin mRNA was expressed in atrial tissue with right atria greater than left atria, with no detectable expression in left ventricle, right ventricle, or skeletal muscle. Beta actin was expressed in all four cardiac chambers and skeletal muscle, and was used to normalize signals. Cardiac expression of the genes for renin and angiotensinogen during embryogenesis suggests that the renin-angiotensin system may be involved in the growth and development of the myocardium.

Angiotensin II receptor-mediated stimulation of cytosolic-free calcium and inositol phosphates in chick myocytes.

We have described previously positive inotropy and increased levels of inositol-l-phosphate as in vitro responses to angiotensin II in cardiac tissue. In this study, changes in cardiac myocyte-free cytosolic calcium stimulated by angiotensin II were monitored with the fluorescent calcium indicator dye Fura-2. There was an initial peak transient increase followed by a sustained increase in cytosolic-free calcium in response to angiotensin II (10(-9)-10(-6) M). The peak transient response in cytosolic-free calcium after addition of angiotensin II (10(-7) M) occurred at 23 +/- 4 sec and was stimulated 2.16-fold (332 +/- 56 nM) above basal levels (154 +/- 14.7 nM). The calcium response was blocked or reversed by addition of verapamil (10(-8) M), lanthanum (0.2 mM) and zero calcium buffer. Angiotensin II receptor-mediated stimulation of inositol phosphates was quantified after separation by high-performance liquid chromatography in cultured chick heart cells prelabeled with L-myo-[1,2-3H(N)]inositol. A time course indicated that the peak response of the angiotensin II (10(-8) M)-stimulated increase in inositol-1,4,5-trisphosphate was at 30 sec. Angiotensin II (10(-8) M) significantly stimulated inositol-1,4-diphosphate (45%) and inositol-1,4,5-trisphosphate (78%) above basal levels. Bordetella pertussis toxin treatment of myocyte cultures in doses (500 ng/ml, 24 hr) shown to fully ADP-ribosylate a toxin-sensitive 41 KD alpha-subunit, blocked completely the angiotensin II-stimulated increases in inositol 1,4-diphosphate, inositol-1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. The rise in cytosolic-free calcium in response to angiotensin II was not blocked or inhibited by toxin pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic treatment with verapamil on adrenoceptor-mediated contraction of rabbit aorta.

Rabbits received twice-a-day doses of verapamil for 8 days and, on day 9 they were sacrificed in order to test contractile responses of the aorta in vitro. Isolated rings of aorta received graded doses of KCl, norepinephrine and norepinephrine along with either phentolamine, phenoxybenzamine or additional (acute) verapamil. Treated rings (those from animals that received verapamil chronically) developed significantly less tension (E) in response to depolarizing doses of KCl, but the degree of developed tension was restored and, in fact, enhanced when the aortic tissue was tested after cold storage 24 hr later (day 10). Responses (E) to norepinephrine in verapamil-pretreated rings were less than in controls by amounts that correlated with the daily verapamil dose and these too were enhanced after cold storage for 24 hr. The equilibrium dissociation constant (KA) of norepinephrine for the alpha 1-adrenoceptor, determined by the method of partial receptor blockade, was increased (reduction in affinity) by amounts that also depended on the daily verapamil dose. In contrast to agonist affinity, phentolamine affinity, determined from Schild-plot analysis, was not different from controls, even for the highest daily dosage of verapamil. Stimulus-effect curves were also the same in control and verapamil-pretreated groups, thus suggesting that the chronic treatment did not affect the basic contractile process distal to the receptor. Further, aortae from pretreated rabbits showed more depression to acute verapamil than was seen in controls and this depression in Emax could not be overcome with even high doses of norepinephrine. In addition to the known alpha-blocking and calcium channel blocking actions of verapamil, these data show that chronic verapamil affects norepinephrine, but not phentolamine-receptor affinity, thus suggesting that the chronic action of this calcium entry blocker may preferentially alter a site on the receptor to which norepinephrine binds.

Measurement of thermodynamic parameters for norepinephrine contraction of isolated rabbit thoracic aorta.

The thermodynamic quantities of change in free energy (delta G degree'), change in enthalpy (delta H degree') and change in entropy (delta S degree') were determined for the interaction of norepinephrine with the alpha-1 adrenoceptor of vascular smooth muscle. Specifically, a standard isolated rabbit thoracic-aorta preparation was used to examine the effect of temperature on norepinephrine-induced isometric tension development. Dissociation constants (KA) for norepinephrine were determined at several temperatures over the range 25-40 degrees C from equiactive concentrations obtained before (A) and after (A') partial irreversible receptor blockade by phenoxybenzamine, plotted as 1/A against 1/A' (KA = (slope-1)/intercept). The values of KA increased with temperature over the range 25-40 degrees C, indicating that the affinity of norepinephrine for the alpha-1 adrenoceptor is an inverse function of temperature over this range. From these results, the thermodynamic quantities delta H degree' and delta S degree' were determined from a van't Hoff plot of In (KA) against 1/T. The relative magnitudes of the change in enthalpy (delta H degree' = -25.58 kcal mol-1) and the change in entropy (delta S degree' = -0.052 kcal mol-1 deg-1) suggest that the reaction between norepinephrine and the alpha-1 adrenoceptor (delta G degree' = -9.15 kcal mol-1) is enthalpy driven, which is consistent with radioligand binding studies of other adrenoceptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)