Hormonal and pharmacological alteration of angiotensinogen secretion from rat hepatocytes.

Isolated hepatocytes were prepared from rat liver by collagenase perfusion and density gradient centrifugation. The hepatocyte preparation released angiotensinogen at a basal rate of 50-120 pmol/g wet weight per h. Release was linear with time for at least 4 h. Angiotensinogen secretion was reduced in the presence of actinomycin D, and inhibited by cycloheximide, puromycin, colchicine and vinblastine. In the presence of tunicamycin, an inhibitor of N-glycosylation, the secretion of angiotensinogen as well as total protein and albumin secretion were diminished. Hepatocytes from nephrectomized rats exhibit an increased secretion rate of angiotensinogen, whereas total protein secretion was unaltered. Preincubation of hepatocytes with hydrocortisone (0.1 mM) or angiotensin II (10 nM) induced an increase of angiotensinogen release. There was no concomitant increase of total protein or albumin secretion, indicating that these effects are not the expression of a general stimulation of protein synthesis and secretion.

Molecular cloning of the porcine beta-1,2-N-acetylglucosaminyltransferase II gene and assignment to chromosome 1q23-q27.

Glycosyltransferases play an important role in the synthesis of glycoproteins. Here we report the isolation of a brain cDNA coding for 89% of the porcine UDP-N-acetylglucosamine:alpha-6-D-mannoside-beta-1,2-N-acetylglucosaminy ltransferase II (EC 2.4.1.143) (GnTII). The cDNA was used for screening a genomic liver DNA library and isolation of a recombinant lambda FIX II phage containing the complete porcine GnTII gene and upstream and downstream sequences. The beta-1,2-N-acetylglucosaminyltransferase II gene harbours a single exon with an open reading frame of 1338 bp coding for a 446 amino acid protein with a calculated molecular mass of 51.1 kDa. The promoter of the GnTII gene is lacking a TATA-box and shows variable transcription start sites. In the 3'-untranslated region a polymorphic polyadenosine stretch was detected. The porcine GnTII gene contains four polyadenylation sites. PCR analysis of a porcine-rodent hybrid cell panel revealed the chromosomal location of the GnTII gene on SSC 1q23-q27. The mapping data of the cell panel were confirmed by fluorescence in situ hybridization (FISH) on metaphase chromosomes.

A cooperative clinical study of methadyl acetate. I. Three-times-a-week regimen.

This was an open clinical trial of methadyl acetate (LAAM) compared with methadone in the maintenance of 636 heroin addicts who had previously been stabilized on a maintenance regimen of methadone. The starting sample assembled by the 13 cooperating clinics were randomly assigned to continued maintenance on methadone (= 308) or crossed over to methadyl acetate (= 328) for a period of 40 weeks. The starting dose was identical to the previously established dose of methadone, but beginning with the second visit, dosage was flexible. Safety was evaluated by clinical and laboratory observations conducted at four-week intervals throughout the study. Relative efficacy was evaluated by illicit drug use, program retention and attendance, and global staff judgments. It is concluded that methadyl acetate is as safe as methadone and, when given three times a week, is an acceptable and effective maintenance drug for many heroin addicts.

A controlled trial comparing buprenorphine and methadone maintenance in opioid dependence.

BACKGROUND: Buprenorphine is a partial agonist at the mu-opioid receptor that has been proposed as an alternative to traditional full agonist maintenance therapy for the treatment of opioid addiction. We report on a clinical trial in which the relative safety and efficacy of long-term fixed-dose buprenorphine maintenance was examined in comparison to low- and high-dose methadone maintenance. METHODS: Two hundred twenty-five treatment-seeking opioid addicts (46 women, 179 men) were randomly assigned to receive, in a double-blind manner, either 8 mg/d of buprenorphine, 30 mg/d of methadone, or 80 mg/d of methadone maintenance over a 1-year period. Objective and subjective measures of efficacy (urine toxicology, retention, craving, and withdrawal symptoms) were examined at the study midpoint and at termination, and safety data were tabulated over the entire 52-week study period. RESULTS: Patients assigned to high-dose methadone maintenance performed significantly better on measures of retention, opioid use, and opioid craving than either the low-dose methadone or the buprenorphine group at both 26-week and 52-week time points. Performance on these measures was virtually identical between the latter two groups. No serious adverse health effects attributable to buprenorphine were noted. CONCLUSIONS: Buprenorphine maintenance at 8 mg/d appears to be less than optimally efficacious under the conditions of the present study. Continued research is needed to reconcile these findings with the more positive results reported by other investigative groups. There are no apparent health risks associated with long-term buprenorphine maintenance at this dosage.

Foster home characteristics and psychiatric patient outcome. The wisdom of Gheel confirmed.

Even with the emphasis on deinstitutionalization, mental health services are still skewed toward hospital and nursing home care. A relatively untapped resource that is likely to receive more attention is the foster home. We previously demonstrated, in a controlled study of psychiatric patients randomly assigned to foster care or continued hospitalization, that foster care produced better social adjustment within four months. From that study we examine characteristics of foster homes associated with the improvement in social functioning. Improved outcome was related to more children in the homes, fewer boarders, and smaller size. Too much stimulation in the environment, more supervision by foster care sponsors, and more intensive follow-up by social work staff was bad for schizophrenic patients but good for nonschizophrenic patients. Neither the sponsors' tolerance and expectation nor the cost of foster care was related to outcome. The size and composition of homes are important and attention needs to be given to finding an enriched environment that is neither too stimulating nor too sterile for schizophrenic patients.

Methadyl acetate and methadone as maintenance treatments for heroin addicts. A veterans administration cooperative study.

This was a double-blind comparison of methadyl acetate and two dose levels of methadone hydrochloride in the maintenance of 430 street heroin addicts from 12 Veterans Administration hospitals. The starting sample consisted of 146 patients receiving low-dose methadone, 142 patients receiving methadyl acetate. Patients were first given 30 mg of both drugs, and doses were incremented by 10 mg/week until they stabilized at methadyl acetate, 80 mg three times a week, and methadone hydrochloride, 50 mg daily or 100 mg daily. Dosage was fixed for the balance of the 40-week treatment period. Safety was evaluated by clinical and laboratory observations conducted at frequent intervals throughout the study. Relative efficacy was evaluated by illicit drug use, program retention and attendance, and global staff judgments. It is concluded that methadyl acetate is as safe a drug as methadone and that it compares favorably with highdose methoadone in terms of efficacy. Both methyadyl acetate and high-dose methadone appear to be better maintenance regimens than low-dose methadone under the conditions of this study.

Hospital vs community (foster) care for psychiatric patients.

The aim of this study was to determine the effectiveness of foster care preparation and placement. Five hundred seventy-two patients from five hospitals were randomly assigned to foster care preparation (experimentals) or continued hospitalization (controls). They were studied before assignment, at placement of experimental subjects, and four months later regarding social functioning, mood, activity, and overall adjustment. Hospitals averaged two months preparing experimental subjects, resulting in 73% placed in foster care. Little change was observed between referral and placement. However, four months after placement, experimental subjects were significantly improved over controls, particularly in social functioning and adjustment. After four months, 88% of the foster care subjects were in the community. Findings suggest that attention should be given to selection criteria, that lengthy preparation may be unnecessary, and that foster care is superior to hospitalization for patients who cannot return to their own homes.

Day treatment and psychotropic drugs in the aftercare of schizophrenic patients. A Veterans Administration cooperative study.

Schizophrenic patients referred for day treatment at the time of discharge from ten hospitals were randomly assigned to receive day treatment plus drugs or to receive drugs alone. They were tested before assignment and at 6, 12, 18, and 24 months on social functioning, symptoms, and attitudes. Community tenure and costs were also measured. The ten day centers were described on process variables every six months for the four years of the study. Some centers were found to be effective in treating chronic schizophrenic patients and others were not. All centers improved the patients' social functioning. Six of the centers were found to significantly delay relapse, reduce sumptoms, and change some attitudes. Costs for patients in these centers were not significantly different from the group receiving only drugs. More professional staff hours, group therapy, and a high patient turnover treatment philosophy were associated with poor-result centers. More occupational therapy and a sustained nonthreatening environment were more characteristic of successful outcome centers.

Restabilization with methadone after methadyl acetate maintenance.

Sixty-eight heroin addicts maintained for 40 weeks on a regimen of methadyl acetate or methadone hydrochloride in a double-blind study were transferred to a uniform dose of 60 mg of methadone daily at the end of their tenure in the study. They were observed for the ensuing six weeks, during which their daily methadone doses were adjusted according to their clinical needs. Patients were observed for symptoms and signs of discomfort and for the amount of illicit drug use during this period of transition. The results indicate that patients maintained on a regimen of methadyl acetate can be readily restabilized with methadone and that sudden decrease of the methadone dose tends to result in the patient's supplementing with illicit heroin. Conversely, increasing methadone doses resulted in a corresponding reduction in illicit drug use. It is suggested that a chronic covert abstinence syndrome may exist in some patients receiving long-term methadone maintenance therapy, and that while it may contribute to their continued illicit drug use, it may have a different pathophysiologic basis and require different therapeutic considerations.

Regulation of hepatic angiotensinogen synthesis and secretion by steroid hormones.

The regulation of angiotensinogen gene expression by steroid hormones in the rat liver has been examined. In the intact animal, dexamethasone (7 mg/kg ip) and estradiol (7 mg/kg sc) caused an increase in plasma angiotensinogen, which became first apparent after 5 or 9 h, respectively, and resulted in plasma concentrations 4.6- and 1.9-fold higher than in controls at 24 h. These changes were preceded by comparable increases in hepatic angiotensinogen messenger RNA (mRNA). In contrast, dihydrotestosterone (10 mg/kg sc) failed to alter plasma angiotensinogen, although hepatic angiotensinogen mRNA and total RNA were slightly elevated. In isolated hepatocytes exposed to either dexamethasone or estradiol (10 microM each) angiotensinogen mRNA started to increase within less than 1 or 3 h, respectively, followed, with a further time lag of about 2 h, by an increase in secretion rate of angiotensinogen. Dihydrotestosterone (10 and 100 microM) induced a rapid increase in total hepatocyte RNA (1.3-fold) and angiotensinogen mRNA (2-fold) with a peak at 2 h. Surprisingly, angiotensinogen secretion remained either unaltered (10 microM dihydrotestosterone) or even decreased (100 microM dihydrotestosterone). In a hepatoma cell line (FT02B) and a subclone (Fe 33) stably transfected with the human estrogen receptor, dexamethasone and estradiol induced an increase in angiotensinogen mRNA and secretion with the same characteristics as in hepatocytes. In conclusion, in this study a direct effect of estradiol on angiotensinogen mRNA and secretion in hepatocytes could be established, which differs from that of dexamethasone by a delayed onset of action. The observation, both in vivo and in vitro, that dihydrotestosterone induced an increase in total RNA and angiotensinogen mRNA, which is not accompanied by an increased angiotensinogen secretion, cannot be explained at present. This study also demonstrates the usefulness of a hepatoma cell line stably transfected with the estrogen receptor gene for the investigation of estrogen-dependent effects in vitro.

Angiotensinogen: an acute-phase protein?

Angiotensinogen has been assumed to be an acute-phase protein, because some forms of acute inflammation, eg, the injection of lipopolysaccharide or cellite or partial hepatectomy, increased the hepatic synthesis of angiotensinogen. In addition, the well-characterized nephrectomy-induced stimulation of angiotensinogen was thought to represent an acute-phase reaction. To evaluate this hypothesis, we examined changes in angiotensinogen secretion by the isolated perfused rat liver after the systemic administration of turpentine or lipopolysaccharide as well as in response to nephrectomy or sham nephrectomy. Comparison was made with the secretion of two typical acute-phase proteins, alpha 1-acid glycoprotein and alpha 2-macroglobulin, and with the secretion of the negative acute-phase protein albumin. All forms of experimental surgery stimulated the secretion of both control acute-phase proteins several-fold. In contrast, the response of angiotensinogen was not uniform; lipopolysaccharide and bilateral nephrectomy stimulated secretion twofold to threefold, sham nephrectomy had no effect, and turpentine decreased the secretion to 30% of the control level. A similar inhomogeneity was found in an additional experiment performed to analyze the direct effects of interleukin-1 or interleukin-6 on the secretion of angiotensinogen by freshly isolated hepatocytes. Interleukin-6 increased but interleukin-1 decreased the mRNA and secretion of angiotensinogen, whereas both cytokines increased the secretion of both acute-phase proteins. Because of this nonuniform behavior of angiotensinogen, it is premature to classify angiotensinogen as an acute-phase protein until a specific function for angiotensinogen during acute inflammation is known.

Mechanism by which angiotensin II stabilizes messenger RNA for angiotensinogen.

The most important specific regulatory mechanism for hepatic angiotensinogen synthesis and secretion is its stimulation by angiotensin II, the effector peptide of the renin-angiotensin system. In the circulating system, this octapeptide is thought to stimulate hepatic angiotensinogen synthesis through a positive feedback loop. In the present study, we have identified the intracellular mechanisms leading to an increase in angiotensinogen messenger RNA (mRNA) and secretion. In a [3H]uridine-dependent pulse and chase system as well as in hepatocytes in which de novo synthesis of mRNA has been blocked by actinomycin D or 5,6-dichlorobenzimidazole riboside, angiotensin II significantly increased the half-life of angiotensinogen mRNA. In contrast, no effect of angiotensin II on the transcription of angiotensinogen mRNA could be observed in a nuclear run-on assay with nuclei from pretreated hepatocytes, whereas dexamethasone, as a positive control, increased the transcription fivefold to sevenfold. We have isolated a 12-kD protein from the polysomal fraction of isolated hepatocytes, which has an affinity to the nontranslated 3' tail of angiotensinogen mRNA. For in vitro transcription of this mRNA fragment, the DNA sequence coding for the nontranslated 3' tail was excised from the vector pRAG 16 and cloned into the transcription vector pGEM 5zf+. Molecular weight and isoelectric point of the mRNA-binding protein correspond to the parameters of a cytosolic protein that becomes phosphorylated by decreased cyclic AMP concentrations as analyzed in [32P]orthophosphate-loaded hepatocytes. In a cytosolic incubation system in which the polysomal fraction was integrated, the mRNA-binding protein increased the half-life of angiotensinogen mRNA significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Relapse of psychiatric patients in foster care.

The authors examined factors related to relapse of psychiatric patients within 1 year of placement in a foster home. They collected data on 210 male patients who had been hospitalized in VA medical centers who were discharged to foster homes in five states. Slightly fewer schizophrenic than nonschizophrenic patients relapsed, but age, length of hospitalization, number of previous hospitalizations, marital status, education, and income did not predict relapse. There was a suggestion that relapsed schizophrenic patients may be characterized before hospital discharge by hyperactivity, undermedication or drug noncompliance, and fewer social skills.

Angiotensin II stimulates angiotensinogen synthesis in hepatocytes by a pertussis toxin-sensitive mechanism.

The role of intracellular messengers in the stimulatory effect of angiotensin II on angiotensinogen synthesis and secretion in hepatocytes was examined. Angiotensinogen secretion was not influenced by modulators of intracellular calcium (calmidazolium, A 23187, Bay K 8644, methoxamine). In contrast, agents decreasing intracellular cAMP (angiotensin II, guanfacine) stimulated, and those increasing cAMP (isoproterenol, glucagon, forskolin) depressed angiotensinogen secretion. An inverse relationship was also observed between cAMP and angiotensinogen mRNA. Pretreatment of hepatocytes with pertussis toxin abolished the stimulation by angiotensin II. It is concluded that angiotensin II-induced stimulation of angiotensinogen synthesis is initiated by inhibition of adenylate cyclase.

Contribution of a 12 kDa protein to the angiotensin II-induced stabilization of angiotensinogen mRNA: interaction with the 3' untranslated mRNA.

Several authors have shown that angiotensin II stimulates hepatic angiotensinogen synthesis in vivo, ex vivo and in vitro. In previous studies we have demonstrated that this effect of angiotensin II depends mainly on a transient inhibition of adenylyl cyclase and is the consequence of a stabilization of angiotensinogen mRNA. In the present study we describe the isolation of a polysomal 12 kDa protein which, in band shift and cross link assays, shows a specific affinity to the 3' untranslated region (3' UTR) of angiotensinogen mRNA and prevents enzymatic degradation of angiotensinogen mRNA in a cell-free incubation system. [32P]UTP-labelled or unlabelled 3' fragments of angiotensinogen mRNA were synthesized on a transcription vector (pGEM5zf+) into which the corresponding DNA sequence was cloned after restriction from vector pRAG 16. Binding of the 12 kDa protein to the radioactively labelled 3' UTR of angiotensinogen mRNA could be displaced by unlabelled 3' UTR mRNA fragments but not by a renin mRNA of comparable length derived from the coding region. The RNA-binding protein appears to be derived from a higher molecular mass precursor (45 kDa) which is preferentially present under reducing conditions in vitro; the active low molecular mass form is evident in the absence of reducing agents. In a cross link experiment we established that a band shift signal which was obtained in the presence of the 45 kDa protein preparation exclusively depends on RNA binding of the active 12 kDa protein. In addition, a phosphorylation step may be involved in the activation of the 12 kDa protein, since its molecular mass and isoelectric point correlate with proteins which were phosphorylated in response to transient decreases of cAMP (induced by guanfacine or angiotensin II) or in response to a direct inhibition of protein kinase A by the cAMP antagonist Rp-cAMP. The importance of phosphorylation reactions for the stabilization of angiotensinogen mRNA was further assessed in a cell-free incubation system of rat liver parenchymal cells. These studies demonstrated that in the presence of acid phosphatase (1 U/ml) the half-life of angiotensinogen was significantly decreased. In the same incubation system the 12 kDa protein increased the half-life of endogenous as well as of exogenous angiotensinogen mRNA three- to fourfold, while no stabilizing effect was apparent when exogenous angiotensinogen mRNA from which the 3' tail had been deleted was added.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for differences in cultured left ventricular fibroblast populations isolated from spontaneously hypertensive and Wistar-Kyoto rats.

OBJECTIVE: To compare fibroblast populations derived from spontaneously hypertensive rats (SHRLJ) and normotensive Wistar-Kyoto rats (WKYLJ) for angiotensin II receptor binding, gene expression of the AT1 receptor and angiotensinogen, hormone responsiveness and phenotypic changes. METHODS: Fibroblasts were isolated by either collagenase B or collagenase P and grown to confluency in the presence of 10% fetal bovine serum. Angiotensin II receptor binding was assessed under both serum and serum-free conditions. Hormonal treatment of cells was conducted in a serum-free background. The concentrations of AT1 receptor and angiotensinogen messenger RNA (mRNA) were determined by liquid hybridization. Phenotypic changes in fibroblast populations were analysed by visualization of lipid-containing vacuoles (oil red O stain) or of alpha-smooth muscle actin-containing fibres (immunostain). RESULTS: SHRLJ collagenase-B cells grew more slowly and had nearly twofold fewer angiotensin II receptors than WKYLJ cells as measured by both radioligand binding and AT1 mRNA content (SHRLJ 1.34 +/- 0.05 versus WKYLJ 5.94 +/- 0.41 pg mRNA per microgram total RNA) but contained significantly more angiotensinogen mRNA (SHRLJ 147 +/- 12 versus WKYLJ 98 +/- 8 fg mRNA per microgram total RNA). Collagenase-P cells from the two strains exhibited similar binding and growth properties. Collagenase-B fibroblasts also exhibited greater responses to exogenous steroids, including a greater shift towards an adipocyte phenotype, than collagenase-P cells. Exogenous angiotensin II promoted transformation towards a myofibroblast cell type, especially in collagenase-P SHRLJ cells. CONCLUSION: Our results indicate that subsets of fibroblasts that differ in growth rate, angiotensin II receptor binding, AT1 and angiotensinogen mRNA levels, structure and steroid responsiveness may be isolated from the left ventricle. The potential importance of these altered phenotypes to cardiac remodelling and hypertrophy warrants further examination.

Reciprocal translocation t(1;15)(p36.2;p11.2): confirmation of a suggestive cytogenetic diagnosis by in situ hybridization and clinical case report on resulting monosomy (1p).

A newborn girl with generalized muscular hypotonia and minor anomalies was referred for chromosome analysis. Cytogenetic investigation showed a satellite and an Ag-positive NOR on the distal short arm of one chromosome 1, thus indicating an unbalanced translocation involving the short arm of an acrocentric chromosome. The phenotypically normal mother had the same satellited chromosome 1 with Ag-positive NOR. One chromosome 15 was the only acrocentric chromosome in her karyotype lacking recognizable satellites and an Ag-positive NOR. Thus a balanced reciprocal translocation between the short arms of chromosomes 1 and 15 in the mother was suggested. The cytogenetic diagnosis was confirmed by nonradioactive in situ hybridization with the most distal DNA probe on chromosome 1, the probe p1-79, localized at chromosome band 1p36.3. The probe was biotinylated by nick-translation, and detection was done by FITC labelled avidin binding. Hybridization signals were observed on both the mother's normal chromosome 1 and the derivative chromosome 15 but not on her derivative chromosome 1. Consequently, the index patient has an unbalanced karyotype with monosomy (1p36.3). Comparing their clinical reports shows that patients with similar terminal deletions of 1p share several manifestations.

Angiotensin II and dexamethasone regulate angiotensinogen mRNA by different mechanisms.

Angiotensinogen synthesis and secretion in the liver is regulated by glucocorticoids and angiotensin II. In isolated hepatocytes in suspension culture, both dexamethasone and angiotensin II induced an increase in angiotensinogen mRNA (2.5- and 4-fold, respectively) with half maximal stimulation at 20 and 200 nM, respectively. In a nuclear run on assay, transcription of the angiotensinogen gene in nuclei from hepatocytes exposed to angiotensin II was not significantly different from controls, whereas dexamethasone-pretreatment dramatically stimulated angiotensinogen mRNA synthesis. By inhibition of transcription in hepatocytes, as well as in [32P]uridine pulse and chase experiments, angiotensin II was shown to stabilize angiotensinogen mRNA, prolonging the intracellular half-life from 83 to 191 min. In polysomal extracts from hepatocytes, a 12 kDa protein could be identified, that binds to a probe of the 3'-untranslated region (UTR) angiotensinogen mRNA. The binding activity of this protein appears to be higher in hepatocytes exposed to angiotensin II, and to have a stabilizing effect on angiotensinogen mRNA. It is proposed that angiotensin II enhances the binding activity of a 12 kDa protein the 3'-UTR of angiotensinogen mRNA, which results in increased stability and transcription of angiotensinogen mRNA.

Patient perspectives of opiate withdrawal.

The objective of this study was to gather information on some aspects of the opiate withdrawal experience from the perspective of the addict. For the most part, results were consistent with expectation, e.g., the sequence with which symptoms appear during withdrawal. However, there were some unexpected findings: the patients' report of the severity of the various symptoms differed from that of experienced clinicians, patients emphasizing more the psychological symptoms. Withdrawal symptoms experienced while on methadone maintenance are also documented.

Role of calcium channels in oxyhemoglobin-induced apoptosis in endothelial cells.

OBJECT: Oxyhemoglobin (OxyHb) released from hemolysed erythrocytes has been considered to be responsible for cerebral vasospasm after subarachnoid hemorrhage. The authors previously reported that OxyHb produced apoptosis in cultured vascular endothelial cells. The change in intracellular Ca++ homeostasis was expected to be one of the possible mechanisms of the cytotoxic effects of OxyHb. This study was undertaken to investigate the protective effects of Ca++ channel blockers on OxyHb-induced apoptosis. METHODS: Cultured bovine coronary artery and brain microvascular endothelial cells (passages 5-9) were used. A cell density study, immunohistochemical staining, and DNA fragmentation analysis were performed to confirm apoptosis. Various concentrations (1-50 microM) of OxyHb were used for 24- to 72-hour incubations with and without Ca++-channel blockers. Oxyhemoglobin produced cytotoxicity leading to cell detachment from the culture dish in time- and concentration-dependent manners. The highest dose (50 microM) of OxyHb produced cell detachment after a 24-hour incubation, and the lower doses (1-10 microM) produced cell detachment after 48 to 72 hours. Immunohistochemical analysis showed that apoptosis occurred in cells that were still attached to the side of the culture dish after 48 to 72 hours of OxyHb treatment (5 microM). The OxyHb (10 microM) produced DNA ladders at 48 to 72 hours. Three Ca++-channel blockers were used to prevent the toxic effect of OxyHb. The voltage-dependent Ca++-channel blocker nicardipine (1 microM), the voltage-independent Ca++-channel blocker econazole (10 microM), and the inorganic Ca++-channel blocker lanthanum (100 microM) all failed to prevent cell detachment or DNA ladders produced by OxyHb. These results were similar in both cell lines. CONCLUSIONS: Oxyhemoglobin produced apoptotic changes in cultured vascular endothelial cells, and Ca++-channel blockers did not prevent OxyHb-induced apoptosis.