Inhibition of endothelial cell migration by cigarette smoke condensate.

Cigarette smoking is among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanism, however, that links cigarette smoking to an increased incidence of atherosclerosis is poorly understood. Endothelial cell (EC) integrity is critical in preventing vascular lesion formation, and after a loss of EC integrity reendothelialization must be rapid and complete. We therefore investigated whether cigarette smoke affected the ECs ability to migrate or altered the intracellular signals generated during migration. The DMSO-soluble fraction of cigarette smoke condensate (CSC), derived from the standard research cigarette, was tested on cultured ECs (HUVEC) derived from human umbilical vein. The addition of CSC caused a dose-dependent decrease in the ability of EC to migrate as measured over a 24-h time period. Nicotine and cadmium sulfate, two constituents of cigarette smoke, individually or in combination, had no effect on migration. Examination of the tyrosine phosphorylation state of various intracellular proteins by Western blot analysis showed that CSC caused the hyperphosphorylation of a 130-kDa protein. In addition, other intracellular proteins showed changes in their phosphorylation states after CSC addition. These results support the hypothesis that CSC is detrimental to normal EC function in maintaining vascular integrity and suggest that smokers are more likely to develop complications of vascular disease due to delayed or incomplete reendothelialization as a consequence of decreased EC migration.

The migratory response to platelet-derived growth factor of smooth muscle cells isolated from synthetic vascular grafts in a canine model.

OBJECTIVE: Previous studies on smooth muscle cells (SMCs) harvested from implanted synthetic grafts demonstrate increased production of platelet-derived growth factor (PDGF) but decreased proliferative response compared with aortic SMCs. The purpose of this study was to determine the migratory response of graft versus aortic SMCs. METHODS: Thoracoabdominal grafts were implanted in beagles. The SMCs were harvested from the graft and infrarenal aorta. Migration was determined with the use of a razor-scrape assay and computerized image analysis. RESULTS: The mean distance migrated and the number of cells that migrated were greater in graft SMCs at baseline (185 +/- 18 micrometer and 108 +/- 17 cells) compared with aortic cells (110 +/- 10 micrometer and 42 +/- 5 cells)(P <.05). Baseline differences persisted after treatment with antibodies to PDGF. The addition of PDGF (10 ng/mL) resulted in increased migration in both graft (229 +/- 23 micrometer and 146 +/- 20 cells) and aortic SMCs (130 +/- 9 micrometer and 70 +/- 5 cells) compared with baseline (P <.05). The relative increase in response to PDGF was similar between the two groups (P = not significant). CONCLUSIONS: Graft SMCs differ phenotypically from aortic SMCs; they exhibit increased basal migration that is independent of autocrine stimulation by PDGF. In contrast to their blunted proliferative response, graft SMCs have a similar migratory response to PDGF compared with aortic SMCs.

Attenuation of rat diaphragm low-frequency fatigue by vanadate in vitro.

Sodium vanadate inhibits protein tyrosine phosphatases, including in skeletal muscle. Vanadate increases contractile force of airway, vascular and gastrointestinal smooth muscle. The present study tested the hypothesis that vanadate augments skeletal muscle contractility. Rat diaphragm muscle strips (n=26 from 12 animals) were studied in vitro at 37 degrees C. Muscles contracted isometrically while stimulated supramaximally with one of two protocols: 30 min of continuous 0.1 Hz stimulation, or 5 min of intermittent 20 Hz stimulation (duty cycle 0.33). Vanadate (500 microM)-treated muscle strips were compared with untreated muscle. Vanadate did not affect force or isometric twitch kinetics of otherwise quiescent muscle. During prolonged 0.1 Hz stimulation, force of control muscles declined by 17 +/- 4% over 30 min, whereas muscles incubated with vanadate maintained force virtually unchanged. Force over time was significantly greater with than without vanadate (P = 0.03), with values being significantly different during the last 10 min of the 30 min stimulation period. In the absence of vanadate force declined at a rate of approximately 0.6% per min, whereas with vanadate the rate of force decline was less than 0.1% per min (P < 0.02). During intermittent 20 Hz stimulation, the degree of force decline was not affected by vanadate at any time over a course of 5 min. Isometric contractile kinetics were not altered by vanadate during either 0.1 or 20 Hz stimulation. These data suggest that vanadate ameliorates low- but not higher-frequency fatigue in diaphragm, suggesting a role for protein tyrosine phosphorylation in the regulation of muscle fatigue resistance.

HS-142-1, a Novel Non-peptide Antagonist for Atrial Natriuretic Peptide Receptor, Selectively Inhibits Particulate Guanylyl Cyclase and Lowers Cyclic GMP in LLC-PK1 Cells.

HS-142-1, a novel atrial natriuretic peptide (ANP) antagonist isolated from the culture broth of Aureobasidium sp., selectively inhibits ANP-induced cyclic GMP accumulation in porcine kidney epithelial LLC-PK1 cells. At concentrations from 0.1 to 100 µg/ml (= 2.5 × 10(-8) - 2.5 × 10(-5) M, given the mean molecular weight is 4, 000), HS-142-1 prevents intracellular cyclic GMP accumulation initiated by 10(-8) M rat ANP in a dose-dependent manner, but not cyclic GMP accumulation produced by 10(-5) M sodium nitroprusside. HS-142-1 alone has no effects on the basal level of cyclic GMP seen in the absence of ANP. No change of intracellular cyclic AMP was observed upon the treatment of the cells with HS-142-1. Further, the selectivity of HS-142-1 for the guanylyl cyclase-linked receptor was confirmed by affinity labeling studies with bovine adrenocortical membranes. HS-142-1 specifically abolished the labeling of the guanylyl cyclase-linked 135-kDa band in a dose-dependent manner, but not the labeling of the 60-kDa band not coupled to the guanylyl cyclase. These results show that HS-142-1 selectively inhibits ANP-mediated accumulation of cyclic GMP in LLC-PK1 cells through interacting with guanylyl cyclase-linked receptors.

Reconstitution of solubilized V1 vasopressin receptors of human platelets.

We describe the reconstitution of solubilized human platelet arginine vasopressin (AVP) receptors into phospholipid vesicles. Purified platelet plasma membranes enriched in AVP receptors [binding equilibrium dissociation constant (Kd) = 1.87 +/- 0.14 nM, maximum number of binding sites (Bmax) = 261 +/- 10 fmol/mg protein] were solubilized with 20 mM sodium cholate. Phospholipid vesicles made of 10% cholesterol, 20% egg phosphatidylcholine, and 70% egg phosphatidylserine were formed by bath sonication. Solubilized AVP receptors were incorporated into the vesicles while the detergent was removed by filtration through Sephadex G-100. The reconstituted receptors retained a high affinity for [3H]AVP (Kd = 3.19 +/- 0.13 nM, Bmax = 257 +/- 9 fmol/mg). Competition experiments with different AVP analogues confirmed the V1 vascular nature of the reconstituted receptors. Saturation experiments carried out with the agonist [3H]AVP and the V1 antagonist [3H]d(CH2)5Tyr(Me)AVP revealed that agonist binding to the reconstituted receptors was divalent cation dependent, whereas antagonist binding was not. Moreover, the affinity of the agonist [3H]AVP for the reconstituted receptors was modulated by the nonhydrolyzable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), whereas [3H]d(CH2)5Tyr(Me)AVP binding affinity was not. The phospholipid vesicles could be loaded with free fura-2 and displayed an enhanced fluorescence caused by calcium entry after addition of ionomycin. However, stimulation by AVP did not induce an increase of free calcium inside the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and identification of human placental endothelin receptor.

Endothelin-1 (ET-1) receptor was identified on the membranes from human placenta and 66% of original binding activity in the membranes was solubilized with 0.75% (w/v) CHAPS. Binding studies of the solubilized membranes using 125I-ET-1 indicated the presence of a single class of high-affinity binding sites with an apparent Kd of 760 pM and a Bmax of 1.8 pmol/mg of protein. The binding was inhibited by addition of unlabeled ET-1 and ET-3 in dose dependent manner. The Ki values of solubilized membranes were 84 pM for ET-1 and 250 pM for ET-3, whereas particulate membranes had weaker affinities (Ki = 410 pM for ET-1, 2500 pM for ET-3). Calcium channel blockers such as nicardipine, verapamil and diltiazem did not affect the binding of 125I-ET-1. Affinity labeling of the particulate and solubilized membranes with CHAPS revealed a specific binding protein with a Mr of 32,000.

Identification of two types of specific endothelin receptors in rat mesangial cell.

Two types of receptors specific for endothelin were identified using cross-linking technique in cultured rat mesangial cells. The molecular weights of these receptors were approximately 58,000 and 34,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The binding of radioiodinated-endothelin to its receptors was inhibited by excess of unlabeled endothelin, but not by nifedipine, nicardipine, verapamil, diltiazem, angiotensin II or [Arg8]-vasopressin. The endothelin binding proteins were solubilized with 1% digitonin and fractionated under non-denaturing conditions by gel filtration. Two endothelin binding peaks eluted at the positions corresponding to the molecular weights of 65,000 and 43,000. These observations indicate that there are two types of specific endothelin receptors in rat mesangial cells which are distinct from voltage dependent L-type calcium channel.

High and low affinity binding sites for endothelin on cultured rat glomerular mesangial cells.

Endothelin contracts glomerular mesangial cells, thereby influencing glomerular size and filtration rate. Here, we demonstrate the presence of two ET-specific binding sites on cultured rat mesangial cells with Kds of 0.76 and 44.70 nM, and maximal binding capacity (Bmax) values of 6.78 x 10(2) and 27.60 x 10(2) binding sites/cell, respectively. Binding of [125I]-ET was maximal at 120 min at 4 degrees C, stable for the subsequent 60 min, and selective. No competition for binding was observed with greater than 1000-fold concentrations of atrial natriuretic peptide, angiotensin II, arginine vasopressin, nicardipine, or nifedipine. The presence of specific receptors for ET on glomerular mesangial cells suggests a major role for this peptide in the regulation of glomerular filtration rate.

Release of atrial natriuretic factor from heart-lung preparations of inbred Dahl rats.

Isolated heart-lung preparations from hypertensive inbred Dahl salt-hypertension sensitive (S) and normotensive inbred Dahl salt-hypertension resistant (R) rats were perfused using 15% washed rat red blood cells in Krebs-Ringer bicarbonate buffer. Atrial pressures were increased by increasing venous return (preload) or by increasing the arterial resistance (afterload). Increases in preload at a constant afterload produced increases in the right and left atrial pressures equivalent between S and R strains. Atrial natriuretic factor (ANF) release was linearly related to right atrial pressure (RAP) or left atrial pressure (LAP) in either strain, but S released more ANF at each level of preload, and the slope of the line relating ANF release to RAP was significantly greater in S than R. When the heart-lung preparations were subjected to changes in afterload at a constant preload, LAP was significantly increased in R but not in S rats, and concomitantly ANF increased in R but not in S. In the afterload experiments, as in the preload studies, S released more ANF than R for comparable LAP. It is concluded that 1) at any atrial pressure, hearts of hypertensive S rats release more ANF than hearts of normotensive R rats, 2) this strain difference is probably a consequence of hypertension, and 3) the observed relationships between ANF release and atrial pressures support the contention that atrial distention stimulates the release of ANF.

Two distinct forms of receptors for atrial natriuretic factor in bovine adrenocortical cells. Purification, ligand binding, and peptide mapping.

The atrial natriuretic factor (ANF) receptor of bovine adrenal cortex was solubilized with Triton X-100 and purified by sequential chromatography on ANF-(99-126)-agarose, GTP-agarose, and wheat germ agglutinin-Sepharose. Two subtypes of ANF receptors were isolated, both of which showed specific ANF binding, whereas one of the ANF receptor subtypes also possessed significant cyclase activity. Both of the receptors showed high capacities (Bmax = 5.7-6.8 nmol/mg of protein) and high affinities (Kd = 54-68 pM) for ANF-(99-126). The cyclase-free receptor had high affinity (Ki = 150-220 pM) to C-terminal truncated ANF analogs, whereas the cyclase-containing receptor had a much weaker affinity (Ki = 10(6)-10(7) pM). When treated with dithiothreitol, the purified cyclase-containing and cyclase-free ANF receptors migrated as a single band at Mr 135,000 and 62,000, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cyclase-free receptor is not a product derived from the cyclase-containing receptor because (i) two proteins with Mr of 135,000 and 62,000 were specifically labeled with 4-azidobenzoyl 125I-ANF-(102-126) in nonsolubilized intact membranes; (ii) the truncated ANF analogs (10(4) pM) prevented the photolabeling of the 62,000-dalton protein but not that of the 135,000-dalton protein; and (iii) two-dimensional peptide mapping showed more than 90% difference between the profiles of the two purified ANF receptor subtypes. This study provides first direct evidence for the existence of two distinct ANF receptors which are different not only in their pharmacological properties but also in their primary structure.

Partial characterization of atrial natriuretic factor in the hearts of Dahl salt-sensitive and salt-resistant rats.

Atria of adult Dahl salt-sensitive rats contain more atrial natriuretic factor (ANF) than those of Dahl salt-resistant rats, as measured by RIA. This strain difference was not seen at 15 days of age, but was observed at and after 30 days of age. Neither a sodium-deficient diet nor an 8% NaCl diet started as early as 2 weeks of age altered this strain difference, although high dietary NaCl was associated with reduced atrial ANF concentration. In addition, no qualitative differences were found in the S and R ANF precursor, as assessed by 1) in vitro translation of atrial mRNA followed by immunoprecipitation of the ANF precursor and polyacrylamide gel electrophoresis, and 2) fractionation of S and R atrial extracts by reverse phase HPLC. These findings support the view that the S vs. R strain difference in atrial ANF concentration is more likely to arise from genetic control of regulatory phenomena than from alterations in the structural part of the ANF gene.

Purification and characterization of two types of atrial natriuretic factor receptors from bovine adrenal cortex: guanylate cyclase-linked and cyclase-free receptors.

Atrial natriuretic factor (ANF) receptors with and without guanylate cyclase activity were simultaneously purified to apparent homogeneity from bovine adrenal zona glomerulosa cell membrane fractions. The particulate guanylate cyclase which co-purified with the ANF receptor showed one of the highest specific activity reported. The receptors with or without the guanylate cyclase activity showed high affinities to ANF (99-126). The receptor without the cyclase showed a high affinity to truncated ANF analogs, ANF (103-123) and ANF (105-121), whereas the cyclase-linked receptor had a much lower affinity to these analogs. Both of the receptors migrated as a single band with a molecular weight of 135,000 daltons on SDS-gel electrophoresis under non-reducing conditions. The 135,000 daltons band of the receptor without the cyclase was shifted to a 62,000 daltons band under reducing conditions, but the band for the cyclase-linked receptor was not shifted. These results demonstrated the presence of two subtypes of ANF receptor in bovine adrenal cortex and indicate two different modes of intracellular action of ANF.

Atrial natriuretic factor in inbred Dahl salt-sensitive and salt-resistant rats.

The atria of adult Dahl salt-sensitive (S) rats contain more atrial natriuretic factor (ANF) than the atria of adult Dahl salt-resistant (R) rats by both radio-immunoassay and bioassay. This strain difference is not present between 1 and 15 days of age but is present at 30 days of age and persists throughout adulthood. The precursors of ANF immunoprecipitated from in vitro translations of atrial messenger RNA (mRNA) had the same molecular weight in S and R strains. Hypothalamic levels of ANF were similar in S and R. Kidneys of weanling S rats were hyporesponsive to ANF compared with R, but as the S rats became hypertensive with age the situation was reversed, with S rats being hyperresponsive to ANF. Aortic vascular responses to ANF were identical in S and R rats. Plasma ANF was similar in S and R rats on low-salt diet, but increased markedly in S, but not R, on high-salt diet. It is concluded that any genetic defect in the ANF system in Dahl S rats is most likely to involve the kidney response to ANF, and that high plasma ANF in salt-fed rats may be a consequence of volume expansion and hypertension.

Characterization of vasopressin receptors of rat urinary bladder and spleen.

By use of tritiated arginine-8-vasopressin (AVP), vasopressin specific binding sites were detected on Sprague-Dawley rat urinary bladder and spleen. In both tissues, one class of high-affinity binding sites was characterized with an equilibrium dissociation constant of 1.61 +/- 0.22 and 1.91 +/- 0.16 nM and a maximal binding capacity of 155 +/- 5 and 110 +/- 11 fmol/mg of protein, for bladder and spleen, respectively. In both tissues, several experimental arguments suggest that these receptors belong to the V1-vascular type: Highly significant correlations were found between the relative agonistic vasopressor activities of eight AVP agonists and their relative abilities to inhibit [3H]AVP binding to the receptors, whereas no such relationship existed when antidiuretic activities were considered. The same profile was also observed with the antagonistic activities of five AVP antagonists. Moreover, AVP (10(-12)-10(-5) M) did not modify the basal cyclic AMP production in either tissue. As cyclic AMP is known to respond to V2 stimulation, the data suggest that the receptors measured are the V1 type. In Dahl rats the receptor characteristics were modulated by salt diet. More interestingly, the number of spleen vasopressin binding sites was always lower in Dahl salt-resistant animals than in the Dahl salt-sensitive animals receiving either a sodium deficient or a 1% NaCl or an 8% NaCl-containing diet. The exploration of vasopressin receptors regulation should facilitate the comprehension of the role played by AVP in different models of experimental hypertension.

Elevated atrial natriuretic polypeptide in plasma of hypertensive Dahl salt-sensitive rats.

The relationship between circulating atrial natriuretic polypeptide (ANP) and blood pressure was studied in inbred Dahl salt-sensitive (S) and inbred Dahl salt-resistant (R) rats. Two month old S and R rats raised on normal rat chow had only small differences in blood pressure and no difference in plasma ANP levels. In contrast, when 6-month-old rats also raised on normal chow were studied, S had markedly elevated blood pressure and a 4 fold increase in plasma ANP compared to R. Similar strain differences in blood pressure and plasma ANP could be induced in young rats by feeding them diets high in salt. In six week old S and R rats which had been fed high salt diet for 3 weeks the S rats showed higher blood pressure and plasma ANP than R rats. The high plasma ANP levels seen in the hypertensive S rats were interpreted to be a response to hypertension and not a cause of hypertension. There was no qualitative strain difference in the plasma ANP molecule as assessed by reverse phase high pressure liquid chromatography.

Atrial natriuretic factor in Dahl rats. Atrial content and renal and aortic responses.

Inbred Dahl salt-sensitive rats had a higher content of atrial natriuretic factor by bioassay in their atria than did inbred Dahl salt-resistant rats. This finding was true both in young 1- to 2-month-old rats, when blood pressure differences between strains were small, and in 7-month-old rats, when blood pressure differences were marked. Atria from salt-sensitive rats had more atrial natriuretic factor than did atria from salt-resistant rats when the rats were fed either low (0.3% NaCl) or high (8% NaCl) salt diet, but a high salt diet suppressed the atrial content of atrial natriuretic factor equally in both strains. In young, prehypertensive salt-sensitive rats, intravenous injections of atrial natriuretic factor caused significantly less natriuresis and diuresis than in salt-resistant rats (p less than 0.05). As the rats aged and salt-sensitive rats became markedly hypertensive, the strain responses to atrial natriuretic factor were reversed, that is, the salt-sensitive rats became more sensitive to atrial natriuretic factor than did the salt-resistant rats. Aortic vascular smooth muscle response to contraction with KCl was equally inhibited in both strains by atrial extracts or atriopeptin II. Thus, the salt-sensitive rat renal hyporesponsiveness to atrial natriuretic factor was not associated with a generalized hyporesponsiveness of vascular tissue to atrial natriuretic factor. It is argued that salt-sensitive rats could have two defects relating to atrial natriuretic factor, one involving hyporesponsive kidneys and another involving decreased release of atrial natriuretic factor from the atria.

Inhibition of aldosterone production by atrial natriuretic factor.

Extracts of rat atrial muscle lowered basal aldosterone release from rat adrenal glomerulosa cell suspensions, and partially inhibited the stimulation of aldosterone release by adrenocorticotropic hormone (ACTH) and angiotensin II. Atriopeptin I, an atrial peptide with natriuretic, diuretic and smooth muscle relaxant activities, significantly decreased basal aldosterone release at 1 pM concentrations. Also, atriopeptin I decreased the sensitivity of the glomerulosa cells to adrenocorticotropin and angiotensin II. These data suggest that peptides contained in mammalian atria affect sodium excretion not only by a direct effect on the kidney, but also indirectly through inhibition of aldosterone production.

Spontaneous nephrotic syndrome in a genetic rat model.

Advances in our understanding of the mechanisms of proteinuria in humans have depended on a variety of animal models. Most of these have been partially satisfactory because they require pretreatment of the animal with chemicals or toxins or they depend on an aging-related glomerular protein leakiness. The strain in this study was obtained by Koletsky after selective inbreeding of the offspring from a hypertensive Kyoto-Wistar and a normotensive Sprague-Dawley rat. The affected animals appear in 25% of the litters, indicating an autosomal recessive gene, and present with a spontaneous and progressive nephrotic syndrome detected as early as 3-5 weeks and associated with obesity, hypertension, hypoalbuminemia, hypercholesterolemia, and hyperlipidemia. Preliminary morphologic and immunofluorescence studies of their kidneys show progressive glomerular segmental sclerotic lesions and prominent mesangial deposition of IgM, a picture which resembles a steroid-resistant form of idiopathic nephrotic syndrome in humans, namely, focal glomerular sclerosis.