KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer.

KRAS mutations occur in 30-40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apc(flox/flox); LSL-Kras(G12D) and CDX2P-G22Cre;Apc(flox/flox) mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT-PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin-nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin-NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.

Regulation of the slow Ca++ channels of myocardial cells.

Contraction of the heart is regulated by a number of mechanisms, such as neurotransmitters, hormones, autacoids, pH, intracellular ATP, and Ca++ ions. These actions are mediated, at least in part, by actions on the sarcolemmal slow (L-type) Ca++ channels, exerted directly or indirectly. The major mechanisms for the regulation of the slow Ca++ channels of myocardial cells includes the following. cAMP/PK-A phosphorylation stimulates the slow Ca++ channel activity, whereas cGMP/PK-G phosphorylation inhibits. DAG/PK-C phosphorylation and tyrosine kinase phosphorylation are suggested to stimulate the slow Ca++ channel activity. Intracellular application of Gs alpha protein increases the slow Ca++ currents (ICa(L)). Lowering of intracellular ATP inhibits ICa(L). Acidosis and increase in [Ca]i inhibits ICa(L). A number of changes in the Ca++ channels also occur during development and aging. Thus, it appears that the slow Ca++ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors, and thereby control can be exercised over the force of contraction of the heart.

Kinetic mechanism of Na+ channel depression by taurine in guinea pig ventricular myocytes.

To examine effects of taurine on the kinetics of the Na+ channel current (I(Na)), action potentials and whole-cell Na+ currents were recorded from single ventricular myocytes of guinea pigs. Kinetic parameters for the activation and inactivation of I(Na) were determined in accordance with the first-order kinetic model. Changes in the kinetic parameters were assessed before and after taurine exposure (5-50 mM). While taurine at concentrations higher than 10 mM decreased the peak I(Na) by ca. 15%, the agent did not alter the reversal potential and the maximum Na+ conductance (GNa). Taurine shifted the steady-state inactivation (h(infinity)) curve toward the negative potential direction and decreased the slope of h(infinity). Concomitantly, the slope of the steady-state activation (m(infinity)) was also slightly decreased and the rate of inactivation in the large potential region (-40 to -30 mV) slightly increased, whereas the rate of the activation appeared to remain unchanged. It is suggested that taurine alters the surface charge of the membrane and reduces the number of charges moving upon activation and inactivation of channels, thereby reducing I(Na).

[Developmental aspects of electrophysiology in cardiac muscle].

Electrical properties of the cardiac muscles drastically change with development. The changes in the current density of ionic currents of cardiomyocytes are inconsistent among species. In cultured embryonic chick ventricular myocytes, the developmental changes in the fast Na+ channel properties (3- to 17-day-old) are reviewed. The sensitivity to TTX, with a KD as high as 2 nM, remains unchanged. The limiting conductance (GNa) increased by 8-10-fold. The activation kinetics such as the steady-state activation (m infinity) and time constant of activation (tau m) remain unchanged. The voltage-dependence of inactivation kinetics such as the steady state inactivation (h infinity) and time constant (tau h) shift in the hyperpolarizing direction. The window conductance tends to be reduced. On the other hand, the L-type Ca2+ channel is important during the development of rat heart, and also the fe-type current (dihydropyridine-resistant) is important in the fetal stage. In chick embryo cardiomyocytes, the L-type channel exhibits long-lasting opening behavior. The behavior is gradually abolished during development, cAMP-dependent protein kinase enhances the Ca2+ channel current on and after the late fetal/embryonic stage. cGMP-dependent protein kinase markedly inhibits the Ca2+ channel current in the fetal/embryonic stage, compared with adult heart. These changes would play an important role for cardiac functions during development.

Suppression of channel conductance by diacetyl monoxime in guinea pig and embryonic chick cardiomyocytes.

AIM: To examine the effects of diacetyl monoxime (DAM), and putative dephospohrylating agent, on conductance of the cardiac Ca, Na, and K channels. METHODS: The Ca (ICa), Na (INa), and K (IK) currents were recorded in single ventricular myocytes from guinea pigs and chick embryos before and after addition of DAM using the whole-cell voltage-clamp technique. RESULTS: DAM 10 mmol.L-1 reduced rapidly the amplitudes of ICa (by about 30%), INa (by about 25%), and IK (by 25%-50%) without alterations of the voltage-dependence. CONCLUSION: DAM was a channel inhibitor of the unique type having nonselective phosphatase activities.

Action potential duration-stabilizing action of taurine in guinea pig ventricular myocytes.

To examine taurine actions on the rate of repolarization of action potentials (AP), L-type Ca2+ (ICa), late outward K+ (Ik) and the inward rectifier currents as affected by the external Ca2+ concentrations ([Ca2+]o), whole-cell voltage-clamp and current-clamp experiments were conducted in guinea pig ventricular myocytes. At a high (3.6 mM) [Ca2+]o, 10 mM taurine suppressed both ICa and IK, shortened AP duration and decelerated the rate (-dV/dt) of terminal repolarization of AP. In contrast, at a low (0.9 mM) [Ca2+]o, taurine intensified both ICa and IK, lengthened AP duration and accelerated -dV/dt. However, at either [Ca2+]o, the resting membrane potential was slightly hyperpolarized, and the inward rectifier, current examined by the ramp-pulse protocol remained unaffected by taurine. Taurine is suggested to maintain a stable AP duration by altering the inward Ca2+ and IK in the opposite directions, depending on [Ca2+]o. The relevance of the stabilizing action of taurine on the AP duration to its reported anti-arrhythmic efficacies is discussed.

Pb2+ reduces the current from NMDA receptors expressed in Xenopus oocytes.

We compared the effects of Pb2+ on four types of NMDA receptors expressed in Xenopus oocytes. Pb2+ reduced the currents evoked by glutamate and glycine. The Ki values of the receptors, epsilon 1/zeta 1, epsilon 2/zeta 1, epsilon 3/zeta 1 and epsilon 4/zeta 1, were 39, 34, 54 and 42 microM, respectively, and their Hill coefficients were 0.53, 4.6, 0.52 and 0.37, respectively. The epsilon 2/zeta 1 receptor that was inhibited in the presence of over 30 microM Pb2+ was not recovered to the control level after a Pb2+ washout for over 30 min, suggesting that epsilon 2/zeta 1 is responsible for the chronic Pb2+ intoxication in the nervous system.

Oxime depression of the fast sodium current in myocardial cells.

Effects of diacetyl monoxime on the fast Na+ current were examined by the whole-cell voltage-clamp method in embryonic chick ventricular myocytes. Diacetyl monoxime (10-20 mM) decreased the duration and amplitude of the action potential and depressed the amplitude of the peak fast inward Na+ current by about 25 (10 mM)-45% (20 mM), without affecting other I-V parameters. Neither the activation and inactivation kinetics of the Na+ channels, such as the time to peak current and the time constant of inactivation, nor the steady state characteristics of the inactivation and activation were affected by diacetyl monoxime. It also did not alter the window conductance and the recovery kinetics from inactivation (reactivation). Hence, diacetyl monoxime suppresses the fast Na+ current, without affecting the time-dependent and voltage-dependent kinetics.

Role of the steady-state Na+ channel current in pacemaker depolarizations in young embryonic chick ventricular myocytes.

To assess the age-related changes in kinetic properties of the cardiac Na+ channel, whole-cell voltage-clamp (v-c) experiments were conducted using 3-, 10- and 17-day-old embryonic chick ventricular heart cells. In line with the first-order kinetic model, kinetic parameters for the activation and inactivation of the channel were determined from the v-c results. Simulation studies using kinetic parameters so determined have reproduced the current-voltage relations and the steady-state inactivation characteristics observed in cells in the three age groups. The rate of depolarization of the simulated action potentials was also comparable to that experimentally recorded. In conclusion, the steady-state Na+ conductance can play a significant role in the automatic depolarizations observed in young embryonic ventricular cells.

Developmental change in fast Na channel properties in embryonic chick ventricular heart cells.

To assess development changes in kinetic properties of the cardiac sodium current, whole-cell voltage-clamp experiments were conducted using 3-, 10-, and 17-day-old embryonic chick ventricular heart cells. Experimental data were quantified according to the Hodgkin-Huxley model. While the Na current density, as examined by the maximal conductance, drastically increased (six- to seven-fold) with development, other current - voltage parameters remained unchanged. Whereas the activation time constant and the steady-state activation characteristics were comparable among the three age groups, the voltage dependence of the inactivation time constant and the steady-state inactivation underwent a shift in the voltage dependence toward negative potentials during embryonic development. Consequently, the steady-state (window current) conductance, which was sufficient to induce automatic activity in the young embryos, was progressively reduced with age.

[Gating properties of cardiac Na channels: from the macroscopic Na current viewpoint].

The sodium channel plays essential roles in the initiation and propagation of action potentials (APs) in excitable tissues including the heart, nerves, and muscles. Na channels in these tissues undergo so-called activation and then inactivation upon step-depolarizations of the cell membrane. Hodgkin and Huxley, early in the 1950s, proposed a mathematical model to describe such events, which was based on voltage-clamp (V-C) data on axonal membranes. However, for the next 30 years or so since the pioneering work of the above workers, electrophysiological studies of the Na channel kinetics in the heart had relied exclusively on AP data (Vmax) as an indirect measure of the Na current instead of V-C data due to difficulty in determining V-C from the complex geometry of cardiac tissues. However, recent development of an isolation procedure for preparing single heart cells and the use of single patch-pipettes for high resolution V-C experiments on these cells have made direct recording of Na channel currents also possible in the heart. Voltage-clamp studies carried out for the last decade have provided several lines of evidence supporting the view that the Na channel properties in the heart of any animal species are somehow more complex than in the axonal membrane and hence showing that Hodgkin-Huxley model can not be directly applied to describe the Na channel behavior in the former type of tissues. Here, we review recent results from V-C studies on Na channel properties with special reference to the macroscopic Na current in cardiac tissues.

Ontogenesis of transmembrane signaling systems for control of cardiac Ca2+ channels.

Cardiac ICa is primarily controlled by the Ca2+ influx across the cell membrane during the action potential. Neurotransmitters and hormones are known to play an important role in controlling the availability of cardiac Ca2+ slow channels and [Ca]i through the transmembrane signaling systems, such as the receptor-adenylate cyclase system and the PI hydrolysis system. This article has reviewed some of the important properties of the transmembrane signaling systems for the control of Ca2+ channels and how they changed during development, especially in the rat heart. Since the phosphorylation hypothesis was first proposed over 15 years ago, considerable advances have been made in understanding the mechanisms for regulation of Ca2+ slow channels at the molecular level. During development, the properties of the cardiac Ca2+ channels, as well as the transmembrane signaling systems, change electrophysiologically, pharmacologically, and biochemically. Because conflicting data have been reported on the developmental changes in these properties, it is still not clear how the complex physiological function of the Ca2+ channels is gradually integrated during development. To answer this question, more data, new concepts, and new approaches are required.

Taurine modulates ion influx through cardiac Ca2+ channels.

The effects of taurine on the inward Ca2+ current (ICa) were investigated by means of the whole-cell voltage-clamp technique in isolated single guinea pig ventricular myocytes. ICa were elicited by 200-ms test pulses from a conditioning holding potential of -45 mV to various test potentials at a rate of 0.5 Hz. Taurine (10-20 mM) had different effects on ICa, depending on the extracellular Ca2+ concentration [( Ca]o). A small stimulatory effect of taurine was found in low [Ca]o (0.8 mM), and a small inhibitory effect was found in high [Ca]o (3.6 mM). Taurine had no significant effect on ICa in normal [Ca]o (1.8 mM). Such dual effects on ICa may explain the various effects reported for taurine especially its dual inotropic actions on cardiac muscle depending upon [Ca]o. Thus, taurine acts in a manner to keep ICa relatively constant. Taurine increased the resting potential irrespective of [Ca]o, suggesting that, in addition, taurine increased K+ conductance and/or ion exchange systems such as the Na/Ca and Na/K exchange.

Developmental changes in beta-adrenergic and cholinergic interactions on calcium-dependent slow action potentials in rat ventricular muscles.

1. Developmental changes in the effect of isoprenaline (Iso) and acetylcholine (ACh) interactions on Ca2(+)-dependent slow action potentials (APs) were studied in the ventricular muscles of foetal (12-20 days post-gestation), neonatal (0-20 days old), and adult (2-3 months old) rats. The slow APs were recorded at 0.2 Hz in partially depolarized preparations (an extracellular K+ concentration of 25 mM). 2. Iso (1 nM to 10 microM) began to increase the Vmax of the slow APs (an approximate indicator of Ca2+ current) on foetal day 18; its potentiating effect became greater with age and reached the adult level about 2 weeks after birth. 3. ACh (10 microM) abolished the Iso (1 microM)-induced increased in the Vmax observed in the late foetal and neonatal periods. 4. The inhibitory effect of ACh on the Vmax was antagonized by atropine but not by pirenzepine, suggesting that ACh reduces Ca2+ current (in the presence of beta-adrenoceptor agonists) by stimulating muscarinic (M2) cholinoceptors. 5. These results suggest that developmental changes in the modulatory effects of beta-adrenoceptor and cholinoceptor agonists on Ca2+ channels occur from a few days before birth to 2 weeks after birth and that the functional coupling between muscarinic cholinoceptors and Ca2+ channels has already been established when the coupling between beta-adrenoceptors and Ca2+ channels starts to operate.

Developmental changes in beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in rat ventricular muscles.

1. In an attempt to explain the previous electrophysiological data on the ontogeny of beta-adrenergic and muscarinic cholinergic interactions on cardiac Ca2+ current, biochemical studies were performed on the ontogeny of beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in cardiac muscle of developing rats: 16-20 days old foetuses, 0-20 days old neonates, and 2-3 months old adults. 2. Developmental changes in cardiac beta-adrenoceptors, muscarinic cholinoceptors, and Ca2+ channels were determined with the use of specific radioligands, [3H]-dihydroalprenolol (DNA), [3H]-quinuclidinyl benzilate (QNB), and [3H]-nitrendipine (NTD), respectively. 3. The Bmax value (fmol mg-1 tissue) for [3H]-DNA binding started to increase on post-gestation day 20, reached almost its maximum level on neonatal day 6, kept almost the same level until neonatal day 20, and then decreased slightly to its adult level. 4. The Bmax value (fmol mg-1 tissue) for [3H]-QNB binding started to increase on post-gestation day 16, reached almost its maximum level on neonatal day 0, remained almost constant until neonatal day 15, and then decreased to its adult level. 5. The Bmax value (fmol mg-1 tissue) for [3H]-NTD binding increased with age between post-gestation day 18 and neonatal day 15, stayed almost constant until neonatal day 20, and then decreased to its adult level. 6. The Kd values for [3H]-DHA, [3H]-QNB, and [3H]-NTD bindings remained almost constant during the developmental period examined. 7. Isoprenaline (Iso) increased the kx of slow action potentials (APs) from post-gestation day 18, and the adult level was reached at about 2 weeks after birth; this developmental time course is similar to that of Ca2+ channels. The number of beta-adrenoceptors also started to increase a few days before birth, but attained its peak about one week earlier than did the Pax of slow APs or the number of Ca2 + channels. 8. Acetylcholine (ACh) almost completely abolished the Iso-induced increase in m,,ax observed from postgestation day 18 to neonatal day 20; this developmental time course for the ACh effect is consistent with the finding that the number of muscarinic cholinoceptors started to increase on post-gestation day 16 and reached a peak on the day of birth. 9. Previous electrophysiological and the present biochemical findings strongly suggest that the functional coupling between muscarinic cholinoceptors and Ca2+ current is already established when the coupling between beta-adrenoceptors and Ca2 + current starts to operate in developing rat hearts.

Effects of lidocaine alone and in combination with tetraethylammonium on the maximum upstroke velocity in guinea-pig papillary muscles.

Effects of 18.5 mumol/l lidocaine on the Vmax in the absence and presence of 10 mmol/l tetraethylammonium chloride (TEA), which specifically prolongs the action potential duration with little effects on Vmax, were studied in guinea-pig papillary muscles in a 10 mmol/l K+ Tyrode solution. The frequency-dependent reduction of Vmax was significantly increased at high frequencies (3 and 4 Hz) in the presence of lidocaine plus TEA. A simulation study was performed under the assumption that lidocaine interacts with open, inactivated and resting sodium channels and that TEA specifically prolongs the inactivated state, and a reasonable fit to the experimental Vmax changes was obtained. There were no significant differences between the effects of lidocaine alone and lidocaine plus TEA on the tonic blocks, time constants and zero-time intercepts of reactivation process of Vmax. The F-test shows that these reactivation processes in all preparations examined are described sufficiently well by monoexponential functions, that is, under the assumption of the linear relationship between available sodium conductance and Vmax. The finding that a nonlinear model does not significantly improve the fit indicates the possibility that the nonlinearity may not be prominent under the present experimental conditions.

Developmental changes in the levels of substrates for cholera toxin-catalyzed and pertussis toxin-catalyzed ADP-ribosylation in rat cardiac cell membranes.

Developmental changes in the substrates for cholera toxin (CTX)- and pertussis toxin (PTX)-catalyzed ADP-ribosylation in cardiac (ventricular) cell membranes were studied in fetal (16- to 20-day), neonatal (0- to 20-day) and adult (2- to 3-month) rats. The CTX and PTX substrates were determined by the method of CTX-catalyzed and PTX-catalyzed ADP-ribosylation of the alpha-subunit of GTP-binding (G) proteins, respectively. As early as fetal day 16, three substrates (45-, 47- and 52-kDa proteins) were identified for CTX-catalyzed ADP-ribosylation and one substrate (41-kDa protein) for PTX-catalyzed ADP-ribosylation. The levels of the three CTX substrates (fmol/mg tissue) increased with development between fetal day 16 and neonatal day 16, and then they decreased to their adult levels. The level of the one PTX substrate (fmol/mg tissue) changed as follows: the substrate decreased between fetal day 16 and the day of birth, increased abruptly for 4 days neonatal and increased slowly thereafter until neonatal day 16, and then decreased to the final adult level. The PTX substrate seems to reach a nearly maximum level earlier than the CTX substrates. This information is essential for understanding the developmental changes in the transmembrane signaling system between membrane receptors and their effectors which are coupled with the stimulatory and inhibitory G proteins.

Fast inward current properties of voltage-clamped ventricular cells of embryonic chick heart.

To determine ionic properties of the fast inward current of embryonic chick hearts, whole cell voltage-clamp experiments were carried out on single cardiomyocytes. Ventricular myocytes trypsin dispersed (spherical, 10-15 microns in diameter, 10.7 pF in membrane capacitance) from 16- to 18-day-old embryonic chick hearts were used. Cells were bathed in solutions containing 140 mM Na+ at room temperature of 22 degrees C. Action potentials recorded via patch electrodes were approximately 103 mV in amplitude, -73 mV in resting potential, and 400 ms in duration. The maximum amplitude of the fast inward-current peak was -1.34 nA (-0.126 mA/microF) and the voltage at which the current was maximum was -29 mV. The reversal potential (Vrev) for this inward current was +35 mV. The inward current was sensitive to change in extracellular Na+ concentration ([Na+]o) (18-mV change in Vrev for halved [Na+]o) and to tetrodotoxin (TTX; 10(-6)-10(-5) M). The inactivation time constant (tau h) and the time-to-peak current decreased from several milliseconds at -60 mV to 1 ms at positive potentials. The steady-state inactivation (h infinity) characteristics showed an S-shaped dependence on voltages between -110 and -60 mV, giving about -90 mV for the half-inactivation voltage. The steady-state activation characteristics (m infinity) also showed sigmoidal voltage dependence, and the half-activation voltage was -54 mV. Both h infinity and m infinity curves crossed each other in the vicinity of -73 mV, giving a maximum window conductance (m infinity 3.h infinity) of 0.00044 at -50 mV. Compared with inactivation, the reactivation of the fast inward channel proceeded much more slowly. It was concluded that except for the low current density of embryonic chick ventricular cells the voltage- and time-dependent kinetics of the fast inward current channel were quite similar to those reported for the fast Na+ channels in single myocytes of adult hearts of other animal species.

Use of single heart cells from chick embryos for the Na+ current measurements.

To record the fast Na+ current, spheroidal heart cells enzymatically-dispersed from 3 approximately 18-day-old chick embryos were used for voltage clamping. The peak of currents in response to voltage steps of 200 ms long from holding potentials of -90 approximately -105 mV were measured. The current-voltage curves for the peak inward current showed U-shaped relations; the averaged peak current of about -1400 pA was observed at about -30 mV and the current reversed sign at +40 approximately +50 mV. Both the peak current and the reversal potential values showed marked [Na]o-dependence, i.e. reduced by 36% and by 20 mV, respectively, for a halved [Na]o. Tetrodotoxin (TTX) partially (10(-6) M) or completely (10(-5) M) suppressed the current. The steady-state inactivation of the current (h infinity) was characterized by the half inactivation voltage of around -80 mV and the slope factor of -4 approximately -8 mV. The half activation voltage and the slope factor for the steady-state activation (m infinity) were -55 mV and 4-6 mV, respectively. The electrophysiological and pharmacological properties were similar between young (3-day-old) and old (15-18-day-old) embryonic heart cells, excepting the much smaller current and the slower onset of TTX action in young embryonic hearts.

Effects of bucumolol, nadolol and nifenalol on maximum upstroke velocity of action potential in guinea pig papillary muscles.

The effects of bucumolol (BUC), nadolol (NAD) and nifenalol (NIF) on contractile forces and on action potentials (APs) were investigated in isolated guinea pig atrial and papillary muscles, respectively. Log 1/ED40 values for the negative inotropic effects of these drugs were 0.097, 10 and 0.74 mmol/l in this order. BUC (50 mumol/l), NAD (0.5 mmol/l) and NIF (0.2 mmol/l) produced about 60, 20 and 20% reduction of Vmax at 1 Hz. The frequency-dependent reductions at these and higher concentrations were greatest for BUC, intermediate for NAD and least for NIF. These potencies at certain frequencies were, as a whole, consistent with log P-potency relationship established in our previous papers (Harada et al. 1981; Ban et al. 1985). The reductions of Vmax in APs in response to premature stimuli during basic stimuli at the rate of 0.25 or 0.027 Hz decayed exponentially during diastolic intervals (DI). The time constants of these decay process (tau) estimated by linear and nonlinear regression analyses and by eye were 12.2-9.6 s for BUC (50-100 mumol/l) and 2.9-4.8 s for NAD (1-2 mmol/l) and 57-87 ms for NIF (0.2-1 mmol/l). In terms of the molecular weight (MW)-log tau relationship (Ban et al. 1985), these tau values are within the 95% fiducial limit for BUC and NAD and deviated from the lower fiducial limit for NIF. The frequency-dependent reductions of Vmax by these drugs were explained in terms of a function of tau and the intercept Ao. Based on the study made by Cohen et al.(ABSTRACT TRUNCATED AT 250 WORDS)