Regulatory effect of castration on endothelins, their receptors and endothelin-converting enzyme in rat seminal vesicle.

OBJECTIVE: To investigate the effects of castration on the expression of endothelins (ETs), ET receptors and ET converting enzyme-1 (ECE-1) in the rat seminal vesicle (RSV). MATERIALS AND METHODS: Sprague-Dawley rats (3 months old) were surgically castrated or sham-operated, and then killed 7 days after surgery. Biochemical and pharmacological properties and the location of ET receptors in the RSV were determined by a series of binding experiments with [125I]ET-1, using membrane particulates and slide-mounted frozen sections of RSV. Expression levels of ETA and ETB receptor subtypes, ET-1, ET-3 and ECE-1 mRNAs were assessed by relative multiplex reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: The density of total ET receptors increased significantly in the seminal vesicle of the castrated rat. The predominance of the ETA receptor subtype in the RSV did not change with castration. Autoradiographic studies showed the presence of ET receptors on the smooth muscle and epithelium of the RSV. In addition, RT-PCR showed an up-regulation in the expression of ETA and ETB receptor subtypes, ET-1 and ECE-1 mRNAs in the seminal vesicle of the castrated rat. However, castration caused no significant change in the expression levels of ET-3 mRNA. CONCLUSION: These findings suggest a regulatory role for testosterone in the expression of the ET receptor system in the RSV.

Progression of heart failure after myocardial infarction in the rat.

This study examined the early neurohumoral events in the progression of congestive heart failure (CHF) after myocardial infarction (MI) in rats. Immediately after MI was induced by coronary artery ligation, rats had severely depressed left ventricular systolic function and increased left ventricular end-diastolic volume (LVEDV). Both left ventricular function and the neurohumoral indicators of CHF underwent dynamic changes over the next 6 wk. LVEDV increased continuously over the study interval, whereas left ventricular stroke volume increased but reached a plateau at 4 wk. Plasma renin activity (PRA), arginine vasopressin, and atrial natriuretic factor all increased, but with differing time courses. PRA declined to a lower steady-state level by 4 wk. Six to 8 wk after MI, CHF rats had enhanced renal sympathetic nerve activity and blunted baroreflex regulation. These findings demonstrate that the early course of heart failure is characterized not by a simple "switching on" of neurohumoral drive, but rather by dynamic fluctuations in neurohumoral regulation that are linked to the process of left ventricular remodeling.

Central mineralocorticoid receptor blockade improves volume regulation and reduces sympathetic drive in heart failure.

The mineralocorticoid (MC) receptor antagonist spironolactone (SL) improves morbidity and mortality in patients with congestive heart failure (CHF). We tested the hypothesis that the central nervous system actions of SL contribute to its beneficial effects. SL (100 ng/h for 28 days) or ethanol vehicle (VEH) was administered intracerebroventricularly or intraperitoneally to rats with CHF induced by coronary artery ligation (CL) and to SHAM-operated controls. The intracerebroventricular SL treatment prevented the increase in sodium appetite and the decreases in sodium and water excretion observed within a week of CL in VEH-treated CHF rats. Intraperitoneal SL also improved volume regulation in the CHF rats, but only after 3 wk of treatment. Four weeks of SL treatment, either intracerebroventricularly or intraperitoneally, ameliorated both the increase in sympathetic drive and the impaired baroreflex function observed in VEH-treated CHF rats. These findings suggest that activation of MC receptors in the central nervous system plays a critical role in the altered volume regulation and augmented sympathetic drive that characterize clinical heart failure.

Neurohumoral regulation in ischemia-induced heart failure. Role of the forebrain.

Congestive heart failure (CHF) is characterized by neurohumoral excitation. Increased sympathetic drive and activation of the reninangiotensin-aldosterone system (RAAS), with vasoconstriction and volume retention, are hallmarks of the CHF syndrome. Treatment strategies have targeted the peripheral influences of these two systems, but have not addressed the central mechanisms that drive them. We monitored the development of CHF following coronary ligation in adult Sprague-Dawley rats. Left ventricular dysfunction characteristic of CHF was confirmed by echocardiography, and the CHF syndrome was validated by measurements of circulating hormones, sodium appetite, thirst, renal sodium and water retention, and renal sympathetic nerve activity (RSNA). In CHF rats, neuronal activity in the hypothalamic paraventricular nucleus (PVN), which mediates downstream effects of forebrain circumventricular organs, was increased and was inhibited by blocking components of the RAAS at the forebrain level. Forebrain (AV3V) lesions and intracarotid (forebrain directed) injections of agents (captopril, losartan, spironolactone) that block RAAS substantially attenuated the behavioral and physiological manifestations of CHF. Intravenous losartan and captopril, in doses that lower arterial pressure, increased RSNA. These findings demonstrate an important role for RAAS-activated forebrain mechanisms in CHF and suggest that the central neural mechanisms driving sympathetic nerve activity and volume retention may persist and promote the progression of CHF despite treatments directed toward the peripheral influences of RAAS.

Cyclooxygenase-2 protein and prostaglandin E(2) production are up-regulated in a rat bladder inflammation model.

Cyclooxygenase-1 and cyclooxygenase-2 mRNAs and proteins and prostaglandin E(2) production are evaluated in a rat model of inflammation in which Escherichia coli lipopolysaccharide is intraperitoneally injected or intravesically instilled into the bladder. While cyclooxygenase-1 mRNA and protein and cyclooxygenase-2 mRNA do not change in bladders treated with lipopolysaccharide, cyclooxygenase-2 protein is elevated in bladders from rats intravesically instilled with lipopolysaccharide or phosphate buffered saline (PBS) or intraperitoneally injected with lipopolysaccharide. Urinary prostaglandin E(2) levels and prostaglandin E(2) synthesis in bladder particulates are elevated by intravesical instillation and intraperitoneal injection of lipopolysaccharide. The nitric oxide donor, S-nitroso-N-acetyl-D,L-penicillamine, increases prostaglandin E(2) synthesis in bladders from lipopolysaccharide intravesically instilled and intraperitoneally injected rats. Lipopolysaccharide increases prostaglandin E(2) synthesis by increasing cyclooxygenase-2 protein levels in rat bladder and prostaglandin E(2) synthesis may be further elevated by increases in nitric oxide caused by an up-regulation of inducible nitric oxide synthase (iNOS).

Urine detection of survivin and diagnosis of bladder cancer.

CONTEXT: Dysregulation of apoptosis may favor onset and progression of cancer and influence response to therapy. Survivin is an inhibitor of apoptosis that is selectively overexpressed in common human cancers, but not in normal tissues, and that correlates with aggressive disease and unfavorable outcomes. OBJECTIVE: To investigate the potential suitability of survivin detection in urine as a novel predictive/prognostic molecular marker of bladder cancer. DESIGN, SETTING, AND PATIENTS: Survey of urine specimens from 5 groups: healthy volunteers (n = 17) and patients with nonneoplastic urinary tract disease (n = 30), genitourinary cancer (n = 30), new-onset or recurrent bladder cancer (n = 46), or treated bladder cancer (n = 35), recruited from 2 New England urology clinics. MAIN OUTCOME MEASURES: Detectable survivin levels, analyzed by a novel detection system and confirmed by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR), in urine samples of the 5 participant groups. RESULTS: Survivin was detected in the urine samples of all 46 patients with new or recurrent bladder cancer using a novel detection system (31 of 31) and RT-PCR (15 of 15) methods. Survivin was not detected in the urine samples of 32 of 35 patients treated for bladder cancer and having negative cystoscopy results. None of the healthy volunteers or patients with prostate, kidney, vaginal, or cervical cancer had detectable survivin in urine samples. Of the 30 patients with nonneoplastic urinary tract disease, survivin was detected in 3 patients who had bladder abnormalities noted using cystoscopy and in 1 patient with an increased prostate-specific antigen level. Patients with low-grade bladder cancer had significantly lower urine survivin levels than patients with carcinoma in situ (P =.002). CONCLUSIONS: Highly sensitive and specific determination of urine survivin appears to provide a simple, noninvasive diagnostic test to identify patients with new or recurrent bladder cancer.

Antioxidant therapy attenuates JNK activation and apoptosis in the remote noninfarcted myocardium after large myocardial infarction.

We hypothesized that the concomitant occurrence of increased oxidative stress, JNK activation, and myocyte apoptosis in the remote myocardium (RM) following a large myocardial infarction (MI) are causally related. Three days following coronary ligation, rats were randomized to treatment with probucol and PDTC (MI-T) or vehicle (MI). Control rats (C) underwent sham operation. At 7 weeks, TBARS assay showed increased level of lipid-peroxidation within the RM in the MI group vs C, which was completely inhibited in the MI-T group. Similarly, Western blot analysis showed a twofold increase in p-JNK in the MI group, vs C, which was attenuated in MI-T, a result confirmed by a JNK-kinase activity. Furthermore, apoptosis was increased within the RM in MI vs C, while this was inhibited in MI-T. We conclude that long-term antioxidant therapy with probucol and PDTC attenuates oxidative stress, JNK activation, and myocyte apoptosis within the RM after large MI.

Regulatory effect of experimental diabetes on the expression of endothelin receptor subtypes and their gene transcripts in the rat adrenal gland.

Endothelins (ETs) mediate paracrine control of vascular tone and secretion of steroids and catecholamines in the adrenal gland through two ET receptor subtypes, ETA and ETB. The differential distribution and function of these subtypes are responsible for the multiplicity of endothelin actions in this tissue. This study examines the regulatory effects of experimental diabetes on the gene expression, subtype specificity and localization of ET receptor subtypes, ET isopeptides, and endothelin-converting enzyme-1 (ECE-1) in the rat adrenal gland. The densities, pharmacological properties and distribution of ET receptor subtypes ETA and ETB in adrenal glands from streptozotocin-induced diabetic, insulin-treated diabetic and age-matched control rats were investigated, using radioligand receptor binding and autoradiographic techniques. The gene expression of ETA and ETB receptors ET-1, ET-3 and ECE-1 was evaluated using relative multiplex reverse transcription/PCR. The induction of diabetes caused a marked reduction in body weight but no significant change in adrenal gland size. The density of ET receptors was significantly increased in the diabetic rat adrenal gland, mainly because of an increase in the expression of ETB receptors. Insulin treatment normalized the diabetes-induced changes in the expression levels of ET receptor subtypes to control levels. The expression level of ET-1 mRNA was up-regulated, whereas ET-3 mRNA was down-regulated in the diabetic adrenal gland compared with the controls. The ECE-1 mRNA level in the adrenal gland was not altered by the induction of diabetes. Autoradiographic studies showed that ETA and ETB are the predominant receptor subtypes in the adrenal medulla and cortex respectively. These results suggest that ETA and ETB receptors are differentially distributed and regulated in the diabetic rat adrenal gland.

Developmental changes in the biochemical and functional properties of endothelin receptors in rabbit renal pelvis.

OBJECTIVE: To examine the effect of age on the biochemical and functional properties, and regional distribution of endothelin (ET) receptors in the rabbit renal pelvis. MATERIALS AND METHODS: The properties of ET receptors in 6-week-old and 6-month-old male rabbit renal pelves were examined using isolated muscle-bath and radioligand receptor-binding techniques. RESULTS: ET-1 caused a significant increase in the contractile force in muscle strips from all regions of the renal pelvis from both age groups, with the following rank order: upper=middle>lower. The magnitude of the ET-1-induced contractile responses were similar in the lower pelvic regions in both ages, but the responses in the upper and middle regions were significantly greater in younger rabbits. ET-1 increased the frequency of spontaneous activity in a concentration-dependent manner in the upper and middle pelvic regions in both age groups, with significantly smaller ED50 values in the younger than in the older rabbits. In both age groups the lower pelvic region lacked spontaneous activity. The density of total ET receptors was higher in the upper and middle regions of the renal pelvis than in the lower renal pelvis of both ages, with the density in the upper and middle regions being greater in older than in younger rabbits. In all regions, ET subtype selective compounds inhibited [125I]ET-1, binding consistent with the predominance of the ETA receptor subtype, except in the lower region of the older rabbits, in which the densities of ETA and ETB subtypes were similar. In all regions, the younger renal pelvis contained a higher proportion of ETA receptors than in older tissues. Light microscopic autoradiographic data indicated the presence of ETA and ETB receptors in smooth muscle and epithelial cells, respectively. CONCLUSION: These data indicate the presence of regional differences in the density of ET receptors and in the contractile responses to ET-1 in rabbit renal pelvis, and that although older rabbit renal pelvis contains more total ET receptors than younger renal pelvis, the latter had a higher portion of the ETA receptor subtype and the younger tissues were more responsive to ET-1.

Expression of endothelin receptor subtypes and their messenger RNAs in diabetic rat prostate: effect of insulin treatment.

Streptozotocin (STZ)-induced diabetes causes an upregulation in the expression of endothelin (ET) receptors in the rat prostate (Eur J Pharmacol 310:197, 1996). We examined the effects of insulin treatment, started 8 weeks after the induction of diabetes, on the expression and distribution of ET receptors and their respective mRNAs in the rat prostate. The densities, pharmacological properties and distribution of ET receptors in the rat prostate were examined using radioligand receptor binding and autoradiographic studies, and gene expression of ET receptors was evaluated utilizing the reverse transcription-polymerase chain reaction (RT-PCR). STZ-injected rats had smaller prostates and reduced serum testosterone levels than control and insulin treated diabetic animals. ET receptor density was shown to be significantly higher in the prostate from diabetic rats than those from either control or insulin treated diabetic animals. The pharmacological profile of prostatic ET receptors was similar in all groups (approximately 80% ET(A); 20% ET(B) subtype). ET receptors were predominantly localized to the prostatic stroma. Induction of diabetes increased the expression of mRNA levels of ET(A) and ET receptors, and insulin treatment reversed this upregulation to control levels. These results indicate that (1) ET receptor subtypes are expressed in the rat prostate as transcription and translation products; (2) insulin can normalize the diabetes-induced upregulation in the expression of ET receptors and their respective mRNAs; and (3) diabetes-induced regression of the prostate may involve an alteration in ET receptors.

Age-related changes in contractile responses of rabbit lower urinary tract to endothelin.

PURPOSE: As there are significant amounts of endothelin (ET) receptors in the mammalian urinary tract, we investigated the pharmacological properties and localization of ET receptors in the rabbit lower urinary tract as a function of age. MATERIALS AND METHODS: The characteristics of ET receptors in bladder dome, trigone and urethra of 6 weeks and 6 months old male rabbits were determined using muscle bath and autoradiographic techniques. RESULTS: ET-1 produces significant contractile responses in smooth muscle strips from bladder dome, trigone, and urethra in both 6 weeks and 6 months old rabbits. Although there was no significant difference in the maximum contractile response of urethral muscle strips to ET-1 between 6 weeks and 6 months old rabbits, the maximum responses to ET-1 were higher in both bladder dome and trigone of 6 weeks than 6 months old rabbits. A selective ETA receptor antagonist, BQ 610, shifted the concentration response curve to ET-1 to the right without decreasing maximal contractile responses in all regions from both age groups, whereas a selective ETB receptor antagonist, IRL 1038, had no significant effect on the contractile response in these tissues. Autoradiographic studies indicate that both ET receptor subtypes are expressed in bladder dome, trigone, and urethra with the ETA subtype being located only in the smooth muscle layers and the ETB subtype being located in both the urothelial and smooth muscle layers. CONCLUSION: Our data indicate the presence of region- and age-dependent differences in the contractile properties of ET receptors in the male rabbit lower urinary tract. Although both ETA and ETB receptor subtypes are present in the smooth muscle layers, the ETA receptor is the sub-type that is primarily involved in the mediation of contractions.

Temporal changes of cytokines and nitric oxide products in urine from renal transplant patients.

BACKGROUND: Acute rejection and urinary tract infection (UTI) both increase nitric oxide synthase (NOS) activity in urine from renal transplant patients. Also, with rejection, a regulatory interplay between nitric oxide (NO) and cytokines has been suggested. Thus, measurement of the temporal changes of NOS products and cytokines in urine will provide a strategy for the diagnosis of acute rejection and for its differentiation from UTI. METHODS: Soluble interleukins (ILs) and NOS-related products, cyclic GMP (cGMP), nitrate, and nitrite were measured in 192 urine samples consecutively collected from 13 patients within the first three months of transplantation. Sixty-seven additional urine specimens were collected randomly from 24 patients for follow-up analysis of the nitrate test. RESULTS: Among patients who experienced rejection, the percentage (%) binding of IL-2 increased within the first five days (P = 0.0004) after transplantation and one to five days prior to the clinical diagnosis (dx) of rejection (P = 0.02). Tumor necrosis factor-alpha, IL-6, and IL-8 increased at the time of rejection dx (P < or = 0.01). With UTI, IL-2 (P = 0.01) decreased one to five days prior to dx, and IL-10 (P = 0.003) increased one to five days after dx. Although cGMP and nitrate are dependent variables, cGMP increased (P < or =0.0009) with both rejection and UTI, and nitrate increased (P = 0.0001) with rejection and decreased (P = 0.0001) with UTI. Prior to formal dx (1 to 5 days), urine nitrate clearly differentiated rejection (3004 to 7451 micromol/L) from UTI (90 to 885 micromol/L) and controls (1059 to 3235 micromol/L). The additional 67 urines demonstrated that the sensitivity of the nitrate test for rejection and UTI was 100%. CONCLUSIONS: In renal transplant patients, specific temporal changes in urine cytokine levels do occur with acute rejection and UTI, but urine nitrate levels are the most precise at differentiating rejection from UTI.

Alterations in G-proteins and beta-adrenergic responsive adenylyl cyclase in rat urinary bladder during aging.

Decreased response of bladder to beta-adrenergic stimulation with aging is related to decreased adenylyl cyclase activity and possibly to changes in guanine nucleotide regulatory protein (G-protein) content or function. G-protein content was quantified by Western blot analysis using antibodies to Gsalpha, Goalpha, and Gialpha in 21-day-old (weanling), 90-day-old (young adult), 6-month-old (adult), and 24-month-old (old) rat bladders. Gi/Go function in bladders with aging was measured by ADP-ribosylation with pertussis toxin. Content of Gsalpha, Goalpha, and Gialpha was lower in 90-day-old bladder than in 21-day-old bladder. Gsalpha content was similar in the 21-day-, 6-month-, and 24-month-old bladders. Gialpha content as well as pertussis toxin-catalyzed ADP-ribosylation was higher in 24-month-old bladders than in 21- and 90-day-old bladders. Pertussis toxin-catalyzed ADP-ribosylation of bladder membranes and treatment of bladder with protein kinase A inhibitors reversed the age-dependent decline in isoproterenol stimulation of adenylyl cyclase. Decreases in beta-adrenergic-induced relaxation response with age in rat bladder are due in part to increases in the content and functional activity of pertussis toxin-sensitive G-protein.

Cardiac hypertrophy is not a required compensatory response to short-term pressure overload.

BACKGROUND: Cardiac hypertrophy is considered a necessary compensatory response to sustained elevations of left ventricular (LV) wall stress. METHODS AND RESULTS: To test this, we inhibited calcineurin with cyclosporine (CsA) in the setting of surgically induced pressure overload in mice and examined in vivo parameters of ventricular volume and function using echocardiography. Normalized heart mass increased 45% by 5 weeks after thoracic aortic banding (TAB; heart weight/body weight, 8.3+/-0.9 mg/g [mean+/-SEM] versus 5. 7+/-0.1 mg/g unbanded, P<0.05). Similar increases were documented in the cell-surface area of isolated LV myocytes. In mice subjected to TAB+CsA treatment, we observed complete inhibition of hypertrophy (heart weight/body weight, 5.2+/-0.3 mg/g at 5 weeks) and myocyte surface area (endocardial and epicardial fractions). The mice tolerated abolition of hypertrophy with no signs of cardiovascular compromise, and 5-week mortality was not different from that of banded mice injected with vehicle (TAB+Veh). Despite abolition of hypertrophy by CsA (LV mass by echo, 83+/-5 mg versus 83+/-2 mg unbanded), chamber size (end-diastolic volume, 33+/-6 microL versus 37+/-1 microL unbanded), and systolic ejection performance (ejection fraction, 97+/-2% versus 97+/-1% unbanded) were normal. LV mass differed significantly in TAB+Veh animals (103+/-5 mg, P<0.05), but chamber volume (end-diastolic volume, 44+/-6 microL), ejection fraction (92+/-2%), and transstenotic pressure gradients (70+/-14 mm Hg in TAB+Veh versus 77+/-11 mm Hg in TAB+CsA) were not different. CONCLUSIONS: In this experimental setting, calcineurin blockade with CsA prevented LV hypertrophy due to pressure overload. TAB mice treated with CsA maintain normal LV size and systolic function.

Experimental diabetes-induced regression of the rat prostate is associated with an increased expression of transforming growth factor-beta.

PURPOSE: Transforming growth factor-beta (TGF-beta), a potent inhibitor of cell growth, plays an important role in the androgen-dependent processes of the prostate through a complex network of growth factors. TGF-beta expression in the prostate is under negative regulatory control of androgen. As experimental diabetes causes a regression of the prostate and decrease in serum testosterone levels in rats, we examined TGF-beta alterations at the mRNA and protein levels in the diabetic rat prostate. MATERIALS AND METHODS: The expression of TGF-beta1 and TGF-beta2 and their respective mRNAs in prostates from streptozotocin (STZ)-induced diabetic, insulin-treated diabetic and age-matched control rats were investigated, using relative multiplex RT-PCR, semi-quantitative Western blotting, and immunohistochemistry. RESULTS: Induction of diabetes caused a significant reduction in prostatic weight and in serum testosterone levels in rats. Both mRNA and protein levels of TGF-beta1, and mRNA level of TGF-beta2 were up-regulated in the diabetic rat prostate. Insulin-treatment normalized changes observed in prostatic weight and serum testosterone levels, and reversed the alterations in the TGF-beta1 and TGF-beta2 expression at the gene transcript and protein levels to control levels. Immunohistochemical studies demonstrated that TGF-beta1 is localized to prostatic stromal cells, whereas TGF-beta2 is located in both epithelial and stromal cells. CONCLUSION: These results suggest that TGF-beta1 and TGF-beta2 may be involved in the diabetes-induced regression of the prostate gland.

Expression and localization of basic fibroblast growth factor in diabetic rat prostate.

OBJECTIVE: To assess fibroblast growth factor-2 (FGF2/bFGF), which is important in the development and maintenance of the normal prostate and in the development of human benign prostatic hyperplasia (BPH) and prostatic carcinoma, in an animal model of experimentally induced diabetes. Materials and methods Using Western blotting and immunohistochemical analyses, the expression of FGF2 in prostates from several groups of rats was investigated. Rats had diabetes for 8 or 16 weeks (induced by intravenous injection with 65 mg/kg streptozotocin); rats were also treated with insulin (starting 8 weeks after the induction of diabetes, for 8 weeks), and two further groups acted as age-matched control rats. Immunohistochemical markers for smooth muscle (alpha-actin) and epithelium (cytokeratin) were used to distinguish different cell types in adjacent prostatic sections. RESULTS: Diabetic rats had smaller prostates and lower serum testosterone levels than their controls; insulin treatment of diabetic rats increased prostatic size and testosterone levels. As shown by Western blotting, diabetes caused greater FGF2 expression than in controls, whereas reverse-transcriptase polymerase chain reaction studies showed similar levels of prostatic FGF-2 mRNA in all groups. Immuno-histochemical studies showed that FGF-2 was expressed in both stromal and epithelial components of the rat prostate. Furthermore, although the expression of FGF2 was higher in epithelial than stromal cells in control prostates, it was distributed uniformly in the diabetic prostate. CONCLUSION: The differences in the level of expression and pattern of distribution of FGF2 suggests a potential role for FGF2 in the changes observed in prostatic growth in diabetic rats.

Antioxidants attenuate myocyte apoptosis in the remote non-infarcted myocardium following large myocardial infarction.

OBJECTIVE: Increased oxidative stress and myocyte apoptosis co-exist in the remote non-infarcted myocardium (RM) following a large myocardial infarction. We proposed that these phenomena are causally related. METHODS AND RESULTS: On day 3 after induction of myocardial infarction, Sprague-Dawley rats were randomized to receive probucol and pyrrolidine dithiocarbamate (MI-T), or vehicle only (MI) for 7 weeks. Control rats (C) received vehicle. At 7 weeks, lipidperoxidation within the RM was assessed by measuring thiobarbituric acid reactive substances, which were significantly increased in MI vs. C, while MI-T was not different from C. There was a significant increase in cardiac myocytes positive for in situ TdT-UTP nick-end labeling within the RM in MI vs. C, which was inhibited in MI-T. Furthermore, internucleosomal DNA fragmentation was clearly demonstrated on agarose gels from RM in the MI group, while it was much less apparent on gels from RM in the C and MI-T groups. Western blot analysis showed a significant increase in p53, Bax and caspase-3 protein expression within the RM of MI vs. C, all of which were inhibited in the MI-T group. Furthermore, there was evidence for an increase in caspase-3 activity within the RM from MI vs. C, which was normalized in the MI-T group. CONCLUSIONS: Long-term treatment with the antioxidants probucol and pyrrolidine dithiocarbamate attenuates oxidative stress, myocyte apoptosis, caspase-3 like activity and the expression of p53, bax and caspase-3 within RM in rats after a large myocardial infarction.

Accuracy of free thyroxine measurements across natural ranges of thyroxine binding to serum proteins.

Systemic inaccuracies, proportional to the concentrations of serum proteins and the thyroxine (T4) they carry, have been reported in direct free T4 immunoassays. However, analytical recoveries of free T4 have not been carefully examined in most current methods, and they have not previously been examined across the pathophysiological range of serum T4 binding. In the present study we investigated ranges of serum T4 binding using free and total T4 measurements from 1359 individuals. Carefully characterized, gravimetrically calibrated, serum-based free T4 test solutions were then prepared with a constant normal free T4 concentration (12 ng/L) and varied serum T4 binding (approximately 300:1 to 24,000:1, ng protein bound T4: ng free T4). These standardized test solutions were analyzed using five T4 analog based free T4 methods. Analytical recoveries were calculated as ratios of actual free T4 measurements to the target value, and expressed as a percent of the target. Analytical recoveries were directly proportional to the extent of serum T4 binding and ranged 2% to 155%, 25% to 131%, 53% to 106%, 37% to 93%, and 37% to 73%, lowest to highest, in different methods. These systemic inaccuracies will confound interpretations of free T4 test results in clinical conditions with altered T4 binding. Future investigations into free T4 status must examine the analytical recovery of the free T4 method(s) used, as they relate to the extent of serum T4 binding in the clinical condition(s) studied.