Methamphetamine-induced toxicity in cultured adult rat cardiomyocytes

In an attempt to differentiate the direct effects of methamphetamine from the indirect sympathomimetic effects on the myocardium, primary culture of adult rat myocytes were established under serum-free conditions, and they were exposed to methamphetamine (1 x 10(-5) and 1 x 10(-3) M) for 1 to 24 h in the presence and absence of 1 x 10(-6) M propranolol. Cardiotoxicity was evaluated by light and ultramicroscopy, release of cytoplasmic enzymes (Lactate dehydrogenase: LDH and Creatine phosphokinase: CPK) and change in membrane permeability (Trypan blue stain). After 24 h methamphetamine treatment, light microscopy exhibited cellular granulation and swelling, myocyte hypercontraction, broken cellular membrane and cellular destruction. After the same time, electron microscopy revealed swelling and irregular mitochondria with disrupted cristaes, clump of sarcomeres with nearly complete loss of organized contractile elements, injury of intracellular membrane system and dissolution of myofibrils. These injurious features were more severe with the 1 x 10(-3) M methamphetamine. Propranolol (1 x 10(-6) M), a beta-adrenergic antagonist, failed to protect the myocytes against methamphetamine-induced cell injury. Release of LDH from methamphetamine (1 x 10(-5) and 1 x 10(-3) M)-treated myocytes increased significantly only after 24 h, while significant CPK release was observed in 1 x 10(-3) M methamphetamine-treated myocytes at 4 h. These findings suggest that methamphetamine exerts direct toxic effects on adult rat myocytes rather than indirect ones via receptors, although further experiments on more concentrations of propranolol are required.     

Different behaviour of the non-sarcomeric cytoskeleton in neonatal and adult rat cardiomyocytes

In this study the function of transforming growth factor-beta (TGF-beta) in preimplantation mouse embryos was examined. By RT-PCR, mRNA for the signalling type I (T beta R-I) and type II (T beta R-II) receptors for TGF-beta was shown to be present in two distinct time windows: in fertilized oocytes and at the blastocyst stage. The function of TGF-beta at these times was analysed in two ways. Firstly, the TGF-beta signalling pathway was blocked by injecting a DNA construct encoding a truncated T beta R-II, that acts as a dominant-negative receptor, in fertilized oocytes, and the effect on development was determined. Secondly, inner cell masses isolated at the blastocyst stage were cultured in vitro with and without TGF-beta under conditions that favour the outgrowth of parietal endoderm. The results show that TGF-beta signalling mediated by maternally expressed receptors is important for development of preimplantation embryos beyond the two-cell stage, and suggest a regulatory role for TGF-beta in the outgrowth of parietal endoderm.     

Triiodothyronine restricts myofibrillar growth and enhances beating frequency in cultured adult rat cardiomyocytes

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.     

Acute effects of morphine on rat intestinal motility

The cumulative urinary excretion over 24 h of pentazocine, under conditions of acidic urinary pH, has been measured in smokers and non-smokers using both male and female subjects (seventy subjects in total). A restricted urban population was studied. An overall three-fold inter-subject variation in elimination was observed. The cumulative urinary excretion of pentazocine was normally distributed in both smokers and non-smokers. Smokers metabolize 40% more pentazocine than non-smokers. It is concluded that induction is principally responsible for the observed subject variability.     

Divergence of beta-myosin heavy chain (betaMHC) expression in fetal rat cardiomyocytes in vitro and adult rat heart in vivo

The myosin heavy chain gene products are an important determinant of myocardial functional properties. Although a strong positive element (beta f1) within the betaMHC promoter region has previously been identified, to date no species comparisons in promoter strength have been made. To examine this question, we have used betaMHC deletion constructs, containing the rat or human beta f1 enhancer region, to determine expression both in vitro using rat fetal cardiomyocytes and in vivo by direct injection into adult rat heart. When reporter constructs were transfected into cultured fetal rat cardiomyocytes, the human beta reporter was expressed more than 3 fold above the equivalent rat construct. Exchange of the beta f1 enhancer indicated that the human sequence of the beta f1 enhancer was largely responsible. However, these findings were not replicated when the reporters were injected into the adult rat heart. In the adult myocardium the levels of reporter expression were similar for the betaMHC promoter reporters studied. These findings demonstrate a divergence between primary cardiomyocytes maintained in culture and the cardiomyocytes found within the intact adult heart.     

Acute effects of inhaled nitric oxide in adult respiratory distress syndrome

This study evaluated the dose-response effect of inhaled nitric oxide (NO) on gas exchange, haemodynamics, and respiratory mechanics in patients with adult respiratory distress syndrome (ARDS). Of 19 consecutive ARDS patients on mechanical ventilation, eight (42%) responded to a test of 10 parts per million (ppm) NO inhalation with a 25% increase in arterial oxygen tension (Pa,O2,) over the baseline value. The eight NO-responders were extensively studied during administration of seven inhaled NO doses: 0.5, 1, 5, 10, 20, 50 and 100 ppm. Pulmonary pressure and pulmonary vascular resistance exhibited a dose-dependent decrease at NO doses of 0.5-5 ppm, with a plateau at higher doses. At all doses, inhaled NO improved O2 exchange via a reduction in venous admixture. On average, the increase in Pa,O2, was maximal at 5 ppm NO. Some patients, however, exhibited maximal improvement in Pa,O2 at 100 ppm NO. In all patients, the increase in arterial O2 content was maximal at 5 ppm NO. The lack of further increase in arterial O2 content above 5 ppm partly depended on an NO-induced increase in methaemoglobin. Respiratory mechanics were not affected by NO inhalation. In conclusion, NO doses < or =5 ppm are effective for optimal treatment both of hypoxaemia and of pulmonary hypertension in adult respiratory distress syndrome. Although NO doses as high as 100 ppm may further increase arterial oxygen tension, this effect may not lead to an improvement in arterial O2 content, due to the NO-induced increase in methaemoglobin. It is important to consider the effect of NO not only on arterial oxygen tension, but also on arterial O2 content for correct management of inhaled nitric oxide therapy.     

Acute effects of catecholamines on function of the rat right heart

OBJECTIVE: Although the haemodynamic effects of catecholamines on the rat left ventricle have been investigated extensively, only few systematic in vivo studies have been performed on the right ventricle. The aim was to examine the acute effects of noradrenaline and isoproterenol on rat right ventricular function. METHODS: Haemodynamic variables were measured during acute, 20 minute infusion of noradrenaline (0.1 mg.kg-1 x h-1) or isoproterenol (12 micrograms.kg-1 x h-1) in female Sprague Dawley rats. To estimate the contribution of alpha and beta receptor stimulation to these effects, eight rats each were infused with prazosin (0.1 mg.kg-1 x h-1), metoprolol (1.0 mg.kg-1 x h-1), or the alpha and beta antagonist carvedilol (0.5 and 1.0 mg.kg-1 x h-1) alone and in combination with noradrenaline or isoproterenol. RESULTS: Noradrenaline and isoproterenol increased right ventricular systolic pressure (RVSP) from 30.3 (SEM 0.5) (n = 32) to 72.7(2.7) (n = 24) and 72.3(4.4) (n = 8) mm Hg, right ventricular (RV) dP/dtmax from 1848(70.3) to 4058(301) and 3612(366) mm Hg.s-1, and heart rate from 329(6) to 371(6) and 420(8) beats.min-1, respectively. Metoprolol completely prevented the isoproterenol induced haemodynamic changes, but neither metoprolol nor prazosin was able to significantly affect the pressure effect of noradrenaline (noradrenaline + metoprolol: 67.3(6.9) mm Hg, noradrenaline + prazosin: 67.0(3.8) mm Hg). The combination of both blockers, however, prevented the noradrenaline induced rise in RVSP (noradrenaline + metoprolol + prazosin: 36.5(5.1), and noradrenaline + prazosin + metoprolol: 30.0(1.2) mm Hg). Carvedilol (1.0 mg.kg-1 x h-1) significantly attenuated the noradrenaline induced RVSP increase (39.1(3.0) mm Hg), but not to the control range. Metoprolol or carvedilol completely prevented the noradrenaline elicited increases in heart rate (254(7) and 287(20) min-1) and RVdP/dtmax, but prazosin alone had no effect on the heart rate and RVdP/dtmax increase. Thus beta receptor blockade alone failed to significantly influence the noradrenaline induced increase of RVSP despite prevention of the increase in heart rate and RVdP/dtmax. Prazosin had a significant effect on RVSP only in combination with metoprolol. CONCLUSIONS: The combined effect of both alpha and beta blockade exceeds the pure addition of the single effects in the rat right ventricle. Moreover, we speculate that the failure to reduce the noradrenaline induced increase in RVSP by either alpha or beta blockade alone is due to the stimulation of the receptor that is not affected by the respective blocker.     

Use of the MTT assay in adult ventricular cardiomyocytes to assess viability: effects of adenosine and potassium on cellular survival

This study used the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay to assess cell viability in isolated quiescent adult guinea-pig ventricular myocytes exposed to different insults or cardioprotective conditions, including adenosine and hyperkalemic-cardioplegia. Optical density (OD), reflecting intracellular reduction of MTT into formazan pigment formation, was a function of the number of viable cells (coefficient of linear correlation approximately 0.99), with MTT reduction preferentially carried out by rod-shaped cardiomyocytes (absorbance at 1.009 +/- 0.013 and 0.006 +/- 0.001 OD units for populations containing 50 and 0% of rod-shaped cells). Following prolonged mechanical (pressure of 1 lb/min for 40 min), chemical (10% DMSO or ethanol) or hypoxic injury (N2-saturated solution), the MTT reductase activity reflected reduction in the number of viable cells by 87%, >50%, and 77%, respectively. In cardiomyocytes exposed to a 40 min hypoxia (with CO2), the MTT reductase activity was 0.056 +/- 0.009 in the absence, and 0.074 +/- 0.008 OD units in the presence of adenosine (1 mM), i.e. adenosine reduced the number of non-viable cells. Also, the MTT assay revealed that the effect of potassium-containing solutions (16 and 32 mM K+) on cellular viability may depend on the extent of insult imposed on cardiomyocytes; i.e. a approximately 24% and 49% increase under mild hypoxia (0.03% CO2), or an 18% decrease in cell viability under severe hypoxia (N2) in pre-injured cells. Thus, the MTT assay used to assess viability of isolated adult cardiomyocytes revealed a direct cytoprotective effect of adenosine and hyperkalemic-cardioplegia by promoting cell survival under certain conditions in vitro.     

Detection of Chlamydia psittaci in enteric subclinical infections in adult sheep, through cell culture isolation

Heretofore, the function of estrogen in the prostate, other than as an antiandrogen, has been unclear. In this review of a growing fund of knowledge about both estrogen and the plasma protein, sex hormone-binding globulin (SHBG), or testosterone-estradiol binding globulin (TeBG), the hypothesis is proposed that estrogen, mediated by SHBG, participates with androgen in setting the pace of prostatic growth and function. It is suggested that the estrogen not only directs stromal proliferation and secretion, but also, through IGF-I, conditions the response of the epithelium to androgen.     

Endotoxin and tumor necrosis factor alpha exert a similar proinflammatory effect in neonatal rat cardiomyocytes, but have different cardiodepressant profiles

Bolus application of endotoxin to healthy volunteers results in reversible hemodynamic alterations, such as observed in septic cardiomyopathy. Currently, endotoxin-induced cardiodepression is mainly attributed to the endotoxin-induced release of proinflammatory cytokines into the circulation, particularly of tumor necrosis factor alpha and interleukin-1, the serum levels of these cytokines being enhanced in sepsis and septic shock, and also in various heart diseases. In this study, we report a proinflammatory effect of endotoxin (1-10 micrograms/ml, 24-h incubation period) on neonatal rat cardiomyocytes in serum-free culture, evidenced by induction of inducible nitric oxide synthase, enhanced release of nitrite (protein synthesis-dependent) and interleukin-6 into the supernatant, as well as an increase in cell-associated interleukin-1 and a specific cardiodepressant profile: endotoxin disrupts beta-adrenoceptor-mediated increase in pulsation amplitude, but alpha-adrenoceptor-induced increase in pulsation amplitude and arrhythmias are not suppressed. In the presence of dexamethasone (0.1 microM), the endotoxin-mediated blockade of beta-adrenergic responsiveness, as well as induction of inducible nitric oxide synthase, enhanced nitrite release and interleukin-1/-6-production are inhibited. In contrast, tumor necrosis factor alpha at a low concentration (10 U/ml) depresses alpha- and beta-adrenergic responsiveness in the presence of dexamethasone in a nitric oxide-independent manner. These data suggest a stimulatory effect of endotoxin on the cardiomyocyte and a specific proinflammatory and nitric oxide-dependent cardiodepressant profile of endotoxin.     

Comparison of the effects of infant handling, isolation, and nonhandling on acoustic startle, prepulse inhibition, locomotion, and HPA activity in the adult rat.

This study examined whether early isolation (EI), early handling (EH), or early nonhandling (NH) in infant rats alters (a) prepulse inhibition (PPI) of the acoustic startle response (ASR) or its disruption by apomorphine, (b) motor activity or its stimulation by amphetamine, or (c) corticosterone activity (because of its modulation of dopamine activity), in adulthood and in comparison with a normal-husbandry postnatal control environment. EI did not affect PPI, reduced PPI disruption by apomorphine in males, and increased amphetamine-stimulated activity in males. NH increased the ASR, reduced activity in the open field, and increased corticosterone reactivity in males. In all paradigms, the effects of EH were similar to those of the control environment. This study provides an important contribution to the evidence on the relationship between postnatal experience and long-term neurobehavioral development in the rat and the relevance of this approach to animal models of neuropsychiatric disorder. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Acute diverticulitis in the young adult

Seven cases of acute diverticulitis in patients 35 years of age or younger are presented. This disease is not rare in young adults. There seems to be a higher incidence in males and an increased need for surgical intervention when younger individuals are affected. The barium enema is useful in confirming the diagnosis.     

Acute sympathectomy by 6-hydroxydopamine in the adult rat: Effects on cardiovascular conditioning and fear retention.

Reports results of an experiment with 38 Charles River male albino rats. Curarized Ss, chemically sympathectomized by 6-hydroxydopamine (6-OH), had lower base-line systolic blood pressures than vehicle-injected controls, but heart rates were equivalent. Maximum blood pressure increase in the vehicle group to unsignaled shock occurred about 2 sec. after shock onset. In the 6-OH group the increase was smaller, reaching maximum 9 sec. after shock. Heart-rate increases to shock were also attenuated by sympathectomy. After 41 paired or random CS and shock presentations, only the paired vehicle group showed a biphasic pressure increase to the CS. Paired and random 6-OH groups showed equivalent long-latency responses to the CS. No consistent group-conditioned heart-rate responses were observed. Vehicle and 6-OH groups did not differ on retention of fear of the CS when tested by a CER procedure 5 days after curarization. (20 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.     

The effects of low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes

The purpose of this study was to examine the effects of low density lipoprotein (LDL) on Ca2+ transients of isolated rabbit cardiomyocytes. Incubation of cardiomyocytes with > or = 1 mg of LDL cholesterol/ml of perfusion medium induced a slow (> or = 30 min) but significant increase (2-fold) in the cellular Ca2+ transient. The time course for the effect was similar to that observed for the accumulation of cholesterol in the cells. Using Dil- labeled LDL as a fluorescent marker for LDL interaction with the cardiomyocytes, it was concluded that LDL interacted via a receptor-mediated event, but probably this was not the primary mechanism whereby the lipid entered the cell. LDL-treated cells were resistant to the depressant actions for ryanodine, nicardipine, and dichlorobenzamil on the cellular Ca2+ transient. Lowering the extracellular Ca2+ concentration removed the stimulatory effect of LDL on the Ca2+ transient. It is concluded that LDL can induce an increase in the magnitude of the Ca2+ transient in isolated cardiomyocytes. This is a relatively slow process. The mechanism appears to involve a stimulation of a transsarcolemmal Ca2+ transport pathway. These findings have important implications for cardiac contractile function in hypercholesterolemic and drug-treated hypercholesterolemic subjects.     

Cocaine-induced effects on isolation stress in neonatal rats.

The effects of cocaine administration on isolation-induced vocalizations and activity levels in 10-day-old rat pups were examined. Day 10 pups given cocaine (1.25, 2.5, 5, 10, and 20 mg/kg ip) vocalized significantly less than their caffeine- (10 mg/kg) and saline-administered siblings during a 5-min isolation period. Cocaine- and caffeine-administered pups also demonstrated a significant increase in overall activity compared with controls. In addition, ip administration of the dopamine antagonist haloperidol (0.5 and 1.0 mg/kg) before 1.25 and 2.5 mg/kg cocaine produced a significant elevation in vocalizations compared with saline pretreatment, which indicates a blocking of cocaine's effect on calling behavior. These results suggest that the endogenous dopamine system involved with reinforcement and reward may quell the stress of isolation in the infant rat. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cocultures of fetal and adult cardiomyocytes yield rhythmically beating rod shaped heart cells from adult rats

Different models of isolated cardiomyocytes are generally used for biochemical, biophysical, and pharmacological studies. Fetal cardiomyocytes can be easily cultured for several weeks regaining their ability for rhythmical and synchronous contractions. For investigations, differentiated myocytes derived from adult hearts are closer to the in situ situation. Unfortunately, these cells at best exhibit irregular and asynchronous contractions at very low frequencies. Already 1 d after seeding calcium-tolerant rod-shaped adult cardiomyocytes on a suitable substrate, the differentiated cells begin to dedifferentiate forming a confluent monolayer. After 7-10 d their beating activities are like those of fetal cells. Therefore, we tried to combine the advantages of both cell types to achieve fully differentiated cardiomyocytes, rod-shaped and rhythmically beating, isolated from adult hearts. Using contractile fetal cells as a substrate for the adult cardiomyocytes, freshly seeded differentiated adult myocytes are paced by the contraction frequency of the fetal monolayer. As a consequence, the rod-shaped adult cardiomyocytes reach frequencies of more than 140 cycles/min without external electrical stimulation. This model enables us to study cardiomyocytes in a state very similar to the in situ situation with respect to morphology, integrity, and contractile behavior.     

Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2.