Physiological role of phosphorylation of the cyclic AMP response element binding protein in rat cardiac nuclei

We determine whether the cyclic AMP signal transduction pathway affects phosphorylation of cyclic AMP response element binding protein and increases muscle gene expression in the heart. Elevation of cyclic AMP results in phosphorylation of the binding protein which is detected using an antibody specific for the phosphorylated, but not the unphosphorylated, form. The protein is present, but not phosphorylated, within the nuclei of myocytes in intact neonatal rat hearts and in high-density cultures. It is not expressed in low-density cultures. Increasing the amount of phosphorylated cyclic AMP with either isoproterenol or forskolin also increases the frequency and force of the beating. The phosphorylated form of the response element binding protein is visible in the nuclei by 10 min and persists for 2 h of drug treatment. A 1.5-fold increase in skeletal α-actin and α-myosin gene expression is detected after 48 h of isoproterenol treatment. However, blockage of beating with a calcium channel blocker (verapamil) in the presence of cyclic AMP results in a similar increased gene expression. This suggests that muscle gene expression can be regulated directly by the cyclic AMP pathway, probably via phosphorylation of the cyclic AMP response element binding protein but independent of contractile activity. Received: 1 December 1995 / Accepted: 2 May 1996     

Frequency-dependent contractile response of isolated cardiac trabeculae under hypo-, normo-, and hyperthermic conditions.

The body is from time to time exposed to nonnormothermic conditions; both hypo- and hyperthermia can occur as a result of external (environment) or internal (pathogens, allergens) stressors. To preserve life under hypo- and hyperthermic conditions, adequate perfusion of vital organs is mandated. Although cardiac output regulation under hyperthermic conditions has been studied, the mechanical response of basic contractile function of the myocardium itself is incompletely understood. Accordingly, we set out to test mechanical output of isolated myocardium under hyperthermic conditions and to compare the results with the hypo- and normothermic response in the same tissue. We observed that, in absence of a frequency change, developed force decreased markedly. At a physiological normal stimulation rate of 6 Hz, developed force decreases to 67.2 +/- 2.6% at 42 degrees C compared with 37 degrees C. In addition, twitch timing characteristics also accelerate, allowing for a faster relaxation; time from peak tension to 50% relaxation is approximately 23% faster (from 31.4 +/- 2.6 to 24.4 +/- 1.7 ms). Although this faster relaxation in turn prevents a steep increase in diastolic tension at high frequencies, the very fast calcium kinetics now prevent a more complete activation of the myofilaments, resulting in a lower twitch-force maximum at hyperthermic conditions. Even at maximal beta-adrenergic stimulation, developed force is well below levels reached at physiological temperature.     

Alterations of cardiac and lymphocyte β-adrenoceptors in rat with chronic heart failure

The alterations of cardiac and lymphocyte β-adrenoceptors were observed in the rats with chronic heart failure produced by constriction of both abdominal aorta and renal artery. The results showed that β₁-adrenoceptor density and mRNA levels were increased, whereas these levels remained unchanged for β₂. The concentrationcontractile response curve for isoproterenol was shifted m the right in cardiac atrium, whereas the concentration-CAMP accumulation response curve for isoproterenol in myocardium was not changed. The number of β-adrenoceptom in blood lymphocyte was markedly reduced. Thus in the heart-failure rats the density of cardiac β-adrenoceptor was increased accompanying reduced β-adrenoceptormediated positive inotropic response, suggesting a post adenylate cyclase dysfunction or impaired contractile components. In contrast, the alteration of β-adrenoceptor in lymphocyte is consistent with the reduced β-adrenoceptor-mediated inotropic response in heart.     

A comparison of the response to induced exercise in red- and white-blooded Antarctic fishes

The physiological responses to forced exercise were studied in yellowbelly and marbled rockcod (Notothenia coriiceps and N. rossii), and the haemoglobinless icefish (Chaenocephalus aceratus), from blood samples obtained via indwelling catheters. The maximal exertion tolerable by N. coriiceps was 3–5 min, although N. rossii was not fully exhausted by this effort, and it proved difficult to elicit sustained maximal activity in C. aceratus. Arterial O₂ tension reflected the relative degree of exhaustion, showing a significant fall in the case of N. coriiceps, little change in N. rossii, and even a rise in C. aceratus as a result of hyperventilation. Such changes in the red-blooded species were not caused by altered O₂ carrying capacity, as there was no change in haematocrit. In Notothenia spp. the decrease in arterial pH was better correlated with a rise in arterial CO₂ tension than with blood lactate concentration, which is reflected in a modest net metabolic acid load. In contrast, the icefish showed an attenuated hypercapnia and a more pronounced lactacidosis, but an insignificant net metabolic acid load. Disturbance in ionoregulation following exercise was limited to an elevated [Cl⁻] in Notothenia, while circulating catecholamine levels remained unusually low in all specimens. The response to stress appears to reflect lifestyle and/or endemic speciation, rather than specific adaptations to the stenothermal environment. Accepted: 9 August 1996     

The effect of age and cholesterol on the rat lung beta-adrenergic system

To assess the influence of membrane lipid composition on beta-adrenergic receptor number and adenylate cyclase activity in aging, we investigated the effect of cholesteryl hemisuccinate on these parameters in lung membranes of 3-, 12-, and 24-month-old CDF (F-344) rats. When cholesteryl hemisuccinate (0.5 mg/ml) was incubated with lung membranes, beta-adrenergic receptor density was increased by 70%. This effect was the same for each age group studied and indicated that the density of both basal and CHS-sensitive receptors is unaltered in rat lung with age. Forskolin, NaF, p[NH]ppG, and isoproteronol-stimulated adenylate cyclase activity is 30% lower in lung membranes from aged rats. Since enzyme activity is affected by the lipid environment and membrane composition often changes with age, we assessed adenylate cyclase activity following cholesteryl hemisuccinate incorporation. There was up to a 75% decrease in adenylate cyclase activity following cholesteryl hemisuccinate incorporation in lung membranes in each of the three age groups. In untreated membranes, there was no significant difference in cholesterol or lipid phosphate content with age. These data suggest that cholesterol content does not account for alterations in senescent rat lung adenylate cyclase activity.     

Basal and ethanol-induced cardiac contractile response in lean and obese zucker rat hearts

Obesity plays a pivotal role in metabolic and cardiovascular diseases. Certain types of obesity may be related to alcohol ingestion, which itself leads to impaired cardiac function. This study analyzed basal and ethanol-induced cardiac contractile response using left-ventricular papillary muscles and myocytes from lean and obese Zucker rats. Contractile properties analyzed include: peak tension development (PTD), peak shortening amplitude (PS), time to PTD/PS (TPT/TPS), time to 90% relaxation/relengthening (RT(90)/TR(90)) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT and +/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes and fluorescence decay time (FDT). In papillary muscles from obese rats, the baseline TPT and RT(90) were significantly prolonged accompanied with low to normal PTD and +/-VT compared to those in lean rats. Muscles from obese hearts also exhibited reduced responsiveness to postrest potentiation, increase in extracellular Ca(2+) concentration, and norepinephrine. By contrast, in isolated myocytes, obesity reduced PS associated with a significant prolonged TR(90), normal TPS and +/-dL/dt. Intracellular Ca(2+) recording revealed decreased resting Ca(2+) levels and prolonged FDT. Acute ethanol exposure (80-640 mg/dl) caused comparable concentration-dependent inhibitions of PTD/PS and DeltaFFI, associated with reduced +/-VT in both groups. Collectively, these results suggest altered cardiac contractile function and unchanged ethanol-induced depression in obesity.     

Characterization of the inflammatory and fibrotic response in a mouse model of cardiac pressure overload

Myocardial fibrosis is an integral component of most cardiac pathologic conditions and contributes to the development of both systolic and diastolic dysfunction. Because of the availability of genetically manipulated animals, mouse models are essential for understanding the mechanisms involved in the pathogenesis of cardiac fibrosis. Accordingly, we characterized the inflammatory and fibrotic response in a mouse model of cardiac pressure overload due to transverse aortic constriction (TAC). Following TAC, mouse hearts exhibited induction of chemokines and proinflammatory cytokines, associated with macrophage, but not neutrophil, infiltration. Induction of inflammatory cytokines was followed by a late upregulation of transforming growth factor (TGF)-β isoforms, activation of the Smad2/3 and Smad1/5 pathways, induction of matricellular proteins, and deposition of collagen. Inflammatory activity decreased after 28 days of TAC; at this timepoint established fibrosis was noted, accompanied by ventricular dilation and systolic dysfunction. Late induction of inhibitory mediators, such as TGF-β, may play an essential role in the transition from inflammation to fibrosis by suppressing inflammatory gene synthesis while inducing matrix deposition. Our findings identify molecular mediators and pathways with a potential role in cardiac fibrosis laying the foundations for studies exploring the pathogenesis of fibrotic cardiac remodeling using genetically targeted mice.     

Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model

Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784–8789). In the present study, two possible intermediates in the synthesis of violaxanthin from β-carotene were identified in P. tricornutum, namely β-cryptoxanthin and β-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light β-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kützing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, β-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms. Received: 16 March 2000 / Accepted: 30 June 2000     

Stretch induced accumulation of total Ca and Na in cytosol and nucleus: a comparison between cardiac trabeculae and isolated myocytes

By means of electron probe microanalysis (EPMA), we quantified changes in total sodium [Na] and calcium [Ca] concentration owing to the following: (i) local axial stretch (LAS) of isolated rat myocytes and (ii) end-to-end stretch (ETES) of rat ventricular trabeculae. For LAS, the distance between patch pipette and a cell-attached stylus was increased by maximally 20%; this activated a nonselective cationic current I(SAC) of approximately -0.5 nA, which was blocked by streptomycin. Trabeculae were stretched end-to-end from 85% L(max) to L(max). Stretch increased cytosolic [Na](total) by 34% in isolated myocytes (p < 0.001) and by 43% in trabeculae (p < 0.001). The increment in nuclear [Na](total) was 21% in myocytes (p < 0.01) and 20% in trabeculae (p < 0.001). Stretch increased [Ca](total) in isolated myocytes, in both cytosol (from 0.63 +/- 0.09 to 1.09 +/- 0.20 mmol/L, p < 0.05) and nucleus (from 0.33 +/- 0.05 to 0.64 +/- 0.13 mmol/L, p < 0.05). In trabeculae, the stretch-induced increment of 51% in cytosolic [Ca](total) remained nonsignificant (p < 0.15). In the nucleus, [Ca](total) did not change. We interpret the difference of stretch on nuclear calcium in myocytes vs. trabeculae with the assumption that LAS, but not ETES, produces shear-stress components that translate the mechanical stimulus deeply into the cell where it may modulate [Ca](total) by signals independent of I(SAC).     

Protective effect of gamma-aminobutyric acid (GABA) against cytotoxicity of ethanol in isolated rat hepatocytes involves modulations in cellular polyamine levels

Summary. Gamma-aminobutyric acid (GABA) is considered to be a multifunctional molecule with various physiological effects throughout the body. It is also evident that the liver contains GABA and its transporter. However, the functions of GABA in liver have not been well documented. In this study, the cytoprotective effect of GABA against ethanol-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. Addition of ethanol induced decrease of cell viability in a dose-dependent manner. However, treatment with GABA resulted in a dose-dependent recovery from ethanol (150 mM)-induced cytotoxicity. GABA reversed the ethanol-induced decrease in intracellular polyamine levels. Furthermore, the addition of polyamines also reversed the ethanol-induced decrease of cell viability. These results suggest that GABA is protective against the cytotoxicity of ethanol in isolated rat hepatocytes and this effect may be modulated by the maintenance of intracellular polyamine levels.     

The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse

Summary. Taurine as well as tauret (retinyliden taurine) levels were measured in locust Locusta migratoria compound eyes. HPLC measurements revealed relatively low taurine levels (1.9 ± 0.16 mM) in dark-adapted eyes. Glutamate, aspartate and glycine levels were 2.0 ± 0.2, 2.7 ± 0.4 and 3.0 ± 0.37 mM, respectively, while GABA was present only in trace amounts. After about 4 h of light adaptation at 1500–2000 lx, amino acid levels in the compound eye were as follows: taurine, 1.8 ± 0.17 mM; glutamate, no change at 2.1 ± 0.2 mM; aspartate sharply increased to 4.7 ± 0.7 mM; glycine slightly decreased to 2.8 ± 0.3 mM; and GABA trace levels. In the compound eye of locust Locusta migratoria, the existence of endogenous tauret in micro-molar range was established. In the dark, levels were several times higher compared with compound eye after light adaptation 1500 lx for 3 h, as estimated by TLC in combination with spectral measurements. Existence of tauret in compound eye is of special interest because in the compound eye, rhodopsin regeneration is based on photoregeneration.     

Prediction and comparison of the secondary structure of legume lectins

Secondary structure prediction for the 4 legume lectins: Concanavalin A, soybean agglutinin, favabean lectin and lentil lectin, was done by the method of Chou and Fasman. This prediction shows that these four lectins fall into a structurally distinct class of proteins, containing high amounts of β-sheet and β-turns. There is a notable similarity in the gross structure of these proteins; all four of them contain about 40–50% of β-sheet, 35–45 % β-turn and 0–10% of α-helix. When the secondary structure of corresponding residues in each pair of these lectins was compared, there was a striking similarity in the Concanavalin A-soybean agglutinin and favabean lectin-lentil lectin pairs, and considerably less similarity in the other pairs, suggesting that these legume lectins have probably evolved in a divergent manner from a common ancestor. A comparison of the predicted potential β-turn sites also supports the hypothesis of divergent evolution in this class of lectins.     

A comparison of the properties of the cardiac beta-adrenergic receptor adenylyl cyclase system in the rat and the marmoset monkey

1. A comparison was made between adrenergic receptor binding properties and catecholamine-stimulated adenylyl cyclase activity in cardiac membrane fractions from the rat and the marmoset monkey. 2. [125I]HEAT and [125I]ICYP were used to determine respectively, the alpha- and beta-adrenergic receptor binding in cardiac membrane fractions. 3. Greatest adrenergic receptor density and degree of specific binding was evident using membranes sedimenting between 6000 and 46,000 g. 4. In rat heart, the ratio of beta- to alpha-adrenergic receptors was 57:43, while for the marmoset this ratio was 92:8. 5. Basal, isoproterenol, sodium fluoride and forskolin-stimulated adenylyl cyclase activities in the rat and marmoset monkey were investigated in several different cardiac membrane fractions. 6. The highest-fold stimulation of adenylyl cyclase activity was present in membranes sedimenting between 0 and 500 g. 7. Adenylyl cyclase activities were higher in the marmoset heart membrane preparations, however the rat heart adenylyl cyclase exhibited greater sensitivity to isoproterenol; ED50 3.8 X 10(-7) M compared with 7.5 X 10(-7) M for the marmoset. 8. Differences between rat and marmoset catecholamine-sensitive adenylyl cyclase activity were apparent when a variety of adrenergic agonists and antagonists were tested. 9. In the marmoset but not the rat, adrenergic antagonists alone stimulated basal adenylyl cyclase activity. 10. Differences in the activation of cardiac adenylyl cyclase by GTP and GMP-PNP were also evident between the rat and the marmoset monkey, particularly with regard to basal and isoproterenol-stimulated activity.     

Effects of D-amino acids on lipoperoxidation in rat liver and kidney mitochondria

Summary. The effects of the amino acids D-ser, D-asp, and D-ala on lipoperoxidation under conditions of hypertension, alcoholism, and ammonemia in rat liver and kidney mitochondria were studied. Under normal conditions, D-alanine increased in 54% free radicals production in liver mitochondria (p < 0.05). The D-amino acids had no effect on kidney mitochondria. D-ser and D-ala increased lipoperoxidation in spontaneously hypertensive rats (SHR) as compared with their normotensive genetic control Wistar-Kyoto (WKY) rats (p < 0.05). During hypertension and in oxidative stress in the presence of calcium, only D-ala produced 46% and 29% free radicals in liver and kidney mitochondria (p < 0.05), respectively. During chronic alcoholism, D-ser increased lipoperoxidation in 80% in kidney mitochondria (p < 0.05), as compared to control. During ammonemia, D-ser produced 41% free radicals.     

Effect of estrogen on calcium-handling proteins, beta-adrenergic receptors, and function in rat heart

Regulation of cellular Ca(2+) cycling is central to myocardial contractile function. Loss of Ca(2+) regulation is associated with cardiac dysfunction and pathology. Estrogen has been shown to modify contractile function and to confer cardioprotection. Therefore, we investigated the effect of estrogen on expression of rat heart myocardial Ca(2+)-handling proteins and beta-adrenergic receptor (beta(1)-AR) and examined functional correlates. Female rats were sham-operated (SHAM) or ovariectomized. Two weeks after ovariectomy rats were injected (i.p.) daily with estradiol benozoate (OVX+EB) or sesame oil (OVX) for 2 weeks. Protein abundance was measured by immunoblotting and mRNA was quantified by real-time RT-PCR. OVX significantly decreased estrogen and progesterone levels and EB replacement returned both estrogen and progesterone to physiological levels. OVX induced a 75% reduction of uterine weight and a gain in body weight. Replacement restored weights to SHAM level. OVX increased and estrogen-replacement normalized abundance of beta(1)-AR and L-type Ca(2+) channel (Cav1.2) protein. OVX decreased sodium-Ca(2+) exchange protein (NCX) and estrogen restored protein abundance to SHAM levels. Sarcoplasmic reticular ATPase (SERCA), phospholamban (PLB), and ryanodine receptor (RyR) abundance was not altered by hormone status. Levels of mRNA encoding for beta(1)-AR, Cav1.2, and NCX were not influenced by OVX or estrogen replacement. OVX had no effect on SERCA and PLB mRNA level but estrogen replacement elicited a significant increase compared to OVX and SHAM. Estrogen-dependent changes in Ca(2+)-handling proteins and beta(1)-AR are theoretically consistent reduced myocellular Ca(2+) load. However, hormone-dependent alterations in protein were not associated with changes in contractile function.     

Monolayer co-culture of rat heart cells and bovine adrenal chromaffin paraneurons

Summary This paper describes a method for the preparation of co-cultures of rat heart cells and bovine adrenal chromaffin paraneurons. The most suitable condition for heart cell isolation was when a combination of paraneurons. The most suitable condition for heart cell isolation was when a combination of trypsin-DNAse I in Locke's solution was used for digestion. The best co-culture conditions were obtained when 10⁶ heart cells were plated on 7- to 8-d-old adrenal chromaffin paraneuron cultures containing 0.5×10⁶ cells per 35-mm diameter culture dishes. Measurements of DNA (heart cells and chromaffin paraneurons), monitoring of beating frequency (heart cells), and catecholamine (chromaffin paraneurons) levels and release indicated that both cell types remain viable and functional, for several weeks. Heart cells started their characteristic contractile activity 24 h earlier when plated either on viable or lysed chromaffin paraneurons, an effect apparently due to faster surface adhesion of heart cells. The beating frequency of heart cells increased after treatment of co-cultures with either noradrenaline or nicotine, with the latter agent acting indirectly through, the release of chromaffin paraneuron catecholamines. Propranolol produced a dose-related inhibition of the responses to either noradrenaline or nicotine, thus suggesting that the increase in myocyte's beating activity was mediated through β-receptors. Anti-myosin and anti-dopamine-β-hydroxylase immunostaining was used for cell type identification and for the demonstration of body-to-body and process-to-process contacts between adrenal chromaffin paraneurons and heart cells. This co-culture system will serve as a starting point of further studies directed to understand a) the influence of a cell type on the development and on the phenotypic characteristics of a second cell type and b) the interaction of cells derived from different organs and species. This study was supported by grant PG-20 from Medical Research Council of Canada and a grant from the Ontario Heart and Stroke Foundation. M. L. N. is a postdoctoral fellow of the MRC.     

Cadmium and heat response of the fungus <Emphasis Type="Italic">Heliscus lugdunensis</Emphasis> isolated from highly polluted and unpolluted areas

Summary. Induction of heat shock protein (Hsp) 70 and distinct metallothionein-like proteins (MTLPs) in response to Cd and heat treatment were studied in two strains of the aquatic hyphomycete Heliscus lugdunensis: Hl-H4, isolated from a heavy metal polluted site, and Hl-BB taken from an unpolluted area. Upon Cd-exposure, Hsp70 was actively synthesized in the strain Hl-H4, and to a much lower degree in the strain Hl-BB. The Hsp70-expression was time- and dose-dependent, reaching a maximum after 24 h incubation with 80 μM Cd. Upon heat-stress, a similar response was observed: a strong Hsp70-expression in Hl-H4, and only a marginal one in Hl-BB. The strains reacted to Cd-exposure by a specific, environmentally related induction of MTLPs, as shown by the highly sensitive bimane derivatisation method of SH-rich proteins. In Hl-H4, a strong expression of 11 kDa MTLP was registered, which followed strictly the induction pattern of Hsp70. This suggests interdependence of the induction mechanisms and roles of these stress proteins in metal resistance. On the contrary, in Hl-BB a weak expression of MTLP of about 20 kDa was observed, exhibiting completely different induction pattern. The results suggest that the specific induction of Hsp70 and/or distinct MTLPs in the range of 11 kDa in H. lugdunensis strain Hl-H4 are essential adaptive mechanisms to continuous heavy metal exposure. Authors’ address: Assoc. Prof. Dr. Konstantin Grancharov, Bulgarian Academy of Sciences, Institute of Molecular Biology, Acad. G. Bonchev Str. Bl. 21, BG-1113 Sofia, Bulgaria