Triiodothyronine Increases Brain Natriuretic Peptide (BNP) Gene Transcription and Amplifies Endothelin-dependent BNP Gene Transcription and Hypertrophy in Neonatal Rat

Brain natriuretic peptide (BNP) gene expression is a well documented marker of hypertrophy in the cardiac myocyte. Triiodothyronine (T 3), the bioactive form of thyroid hormone, triggers a unique form of hypertrophy in cardiac myocytes that accompanies the selective activation or suppression of specific gene targets. In this study, we show that the BNP gene is a target of T 3 action. BNP secretion was increased 6-fold, BNP mRNA levels 3-fold, and BNP promoter activity 3–5-fold following T 3 treatment. This was accompanied by an increase in myocyte size, sarcomeric organization, and protein synthesis. Of note, several of the responses to T 3 synergized with those to the conventional hypertrophic agonist endothelin. The response to the liganded thyroid hormone receptor (TR) was mediated by an unusual thyroid hormon

Synthesis of Rat Myosin Light Chains in Heterokaryons Formed between Undifferentiated Rat Myoblasts

ABSTRACT The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99 % pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin ligh

Steady-State Current-Voltage Relationship of the Na/K Pump in Guinea Pig

ABSTRACT Whole-cell currents were recorded in guinea pig ventricular myocytes at ~36 ~ before, during, and after exposure to maximally effective concentrations of strophanthidin, a cardiotonic steroid and specific inhibitor of the Na/K pump. Wide-tipped pipettes, in combination with a device for exchanging the solution inside the pipette, afforded reasonable control of the ionic composition of the intraceUular solution and of the membrane potential. Internal and external solutions were designed to minimize channel currents and Na/Ca exchange current while sustaining vigorous forward Na/K transport, monitored as strophanthidinsensitive current. 100-ms voltage pulses from the-40 mV holding potential were used to determine steady-state levels of membrane current between-140 and + 60 mV. Control experiments demonstrated that if the Na/K pump cycle were first arrested, e.g., by withdrawal of external K, or of both internal and external Na, then neither strophanthidin nor its vehicle, dimethylsulfoxide, had any discernible effect on steady-state membrane current. Further controls showed that, with the Na/K pump inhibited by strophanthidin, membrane current was insensitiv

Properties of L-Type Calcium Channel Gating Current in Isolated Guinea Pig

ABSTRACT Nonlinear capacitative current (charge movement) was compared to the Ca current (/ca) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from-50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between-40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to indtice "extra " charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltagedependent manner (K D--200 nM at-40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19-+ 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated " by means that do not affect the voltage sensor

Regulation of Rat Myosin Light-Chain Synthesis in Heterokaryons between 5-Bromodeoxyuridine-blocked Rat Myoblasts and Differentiated

ABSTRACT Terminal cell differentiation in a variety of model systems is inhibited by the thymidine analogue 5-bromodeoxyuridine (BUdR). We investigated the mode of action of BUdR by forming heterokaryons between undifferentiated BUdR-blocked rat myoblasts and differentiated chick skeletal myocytes. We analyzed newly synthesized proteins on twodimensional polyacrylamide gels. The induction of rat skeletal myosin light-chain synthesis was reduced fivefold, as compared with controls, when chick myocytes were fused to BUdRblocked rat myoblasts. This indicates that plasma membrane effects cannot be the proximate cause for the inhibition of myogenesis by 8UdR, since BUdR is able to block the effect of chick inducing factors even when a differentiated chick myocyte is in direct cytoplasmic continuity with the BUdR-blocked rat nucleus. The observation that chick cells required an 80 % substitution of BUdR for thymidine to block myogenesis, whereas L6 rat myoblasts required only a 20 % substitution led to a hypothesis involving a DNA-mediated action of BUdR. This model yielded three testable predictions: (a) putative chick inducing molecules should be present in limiting quantities, (b) exploiting genedosage effects to increase the quantity of putative chick inducing factors might overcome th

Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1820/1025935 Mol Pharmacol 62:1482–1491, 2002 Printed in U.S.A. Identification of PKC � Isoform-Specific Effects in Cardiac

Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in multiple cellular processes. In the heart, PKC signaling is involved in hypertrophic agonist-induced gene expression and hypertrophic growth. To investigate the specific function of PKC signaling in regulating cardiomyocyte growth, we used antisense oligonucleotides to inhibit PKC �, the major isozyme present in the neonatal heart. Transfection of cultured neonatal cardiomyocytes with antisense PKC � oligonucleotides resulted in a marked reduction in both PKC � mRNA and protein levels. PKC � antisense treatment also reduced phenylephrine (PE)-induced PKC activity and perinuclear translocation of PKC�. Antisense inhibition of PKC � led to reduction of PE-induced increase in skeletal �-actin mRNA levels and atrial natriuretic peptide (ANP) secretion but had n

Calcium Release From the Sarcoplasmic Reticulum in

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Effects of External Protons on Single Cardiac Sodium Channels from Guinea

ABSTRACT The effects of external protons on single sodium channel currents recorded from cell-attached patches on guinea pig ventricular myocytes were investigated. Extracellular protons reduce single channel current amplitude in a dose-dependent manner, consistent with a simple rapid channel block model where protons bind to a site within the channel with an apparent pKn of 5. I0. The reduction in single channel current amplitude by protons is voltage independent between-70 and-20 mV. Increasing external proton concentration also shifts channel gating parameters to more positive voltages, consistent with previous macroscopic results. Similar voltage shifts are seen in the steady-state inactivation (h®) curve, the time constant for macroscopic current inactivation (%), and the first latency function describing channel activation. As pHo decreases from 7.4 to 5.5, the midpoint of the h ® curve shifts from-107.6 + 2.6 mV (mean-+ SD, n = 16) to-94.3-+ 1.9 mV (n = 3, P < 0.001). These effects on channel gating are consistent with a reduction in negative surface potential due to titration of negative external surface charge. The Gouy-Chapman-Stern surface charge model incorporating specific proton binding provides an excellent fit to the dose-response curve for the shift in the midpoint of the h ® curve with protons, yielding an estimate for total negative surface charge density of-le/490/ ~ and a pK. for proton binding of 5.16. By reducing external surface Na + concentration, titration of negative surface charge can also quantitatively account for the reduction in single Na ÷ channel current amplitude, although we cannot rule out a potential role for channel block. Thus, titration by protons of a single class of negatively charged sites may account for effects on both single channel current amplitude and gating

[Na] and [K] Dependence of the Na/K Pump Current-Voltage Relationship in

ABSTRACT Na/K pump current was determined between- 140 and +60 mV as steady-state, strophanthidin-sensitive, whole-cell current in guinea pig ventricular myocytes, voltage-clamped and internally dialyzed via wide-tipped pipettes. Solutions were designed to minimize all other components of membrane current. A device for exchanging the solution inside the pipette permitted investigation of Na/K pump current-voltage (I-V) relationships at several levels of pipette [Na] ([Na]m) in a single cell; the effects of changes in external [Na] ([Na]o) or external [K] ([K]o) were also studied. At 50 mM [Na]#p, 5.4 mM [K]o, and ~150 mM [Na]o, Na/K pump current was steeply voltage dependent at negative potentials but was approximately constant at positive potentials. Under those conditions, reduction of [Na]o enhanced pump current at negative potentials but had little effect at positive potentials: at zero [Na]o, pump current was only weakly voltage dependent. At 5.4 mM [K]o and ~150 mM [Na]o, reduction of [Na]~, from 50 mM scaled down the sigmoid pump I-V relationship and shifted it slightly to the right (toward more positive potentials). Pump current at 0 mV was activated by [Na]p ~ according t

On the Role of Sodium Ions in the Regulation of the Inward-Rectifying Potassium Conductance in Cat

ABSTRACT The conductance of the inward-rectifying K + current (IK~) in isolated cat ventricular myocytes is decreased by reducing the extracellular Na § concentration. Using a whole-cell patch-clamp technique, possible mechanisms underlying this Na + dependence were investigated. These included (a) block of inward K + current by the Na + substitute, (b) changes in membrane surface charge associated with removal of extraceUular Na § (c) increases of intraceUular Ca ~+ due to suppression of Na-Ca exchange, (d) reduction of a Na+-dependent K + conductance due to a subsequent decrease of intracellular Na +, (e) reduction of IK ~ conductance (gK~) associated with reduction of intracellular pH due to suppression of Naproton exchange. The findings support the hypothesis that the effect of removing Na § is mediated through a decrease in intracellular pH. These include observations that: (a) reducing internal pH by reducing external pH caused a decrease in gK~, and the conductance changes caused by reducing extracellular pH and removing extracellular Na § were not additive: (b) the effect of reducing pHo was attenuated by dialyzing with a low pH internal solution; (c) gK1 was reduced by exposure to the Na-proton exchange inhibitor dimethylamiloride, and this effect was absent in the absence of Na § These findings imply that physiological or pathological processes such as ischemia and metabolic or respiratory acidosis which can produce intracellular acidosis should be expected to affect K + permeation through the IKa channel