Genistein attenuates postischemic depressed myocardial function by increasing myofilament Ca2+ sensitivity in rat myocardium

The present study investigated whether genistein, a broad-spectrum tyrosine kinase inhibitor, could increase the myofilament Ca(2+) sensitivity and partially reverse postischemic depressed myocardial function. Left ventricular papillary muscles were isolated from adult Wistar rats and loaded with the Ca2+ indicator, aequorin. The use of fluorocarbon immersion with hypoxia simulated a model of ischemia. Myofilament responsiveness to Ca2+ was evaluated from force-[Ca2+]i relationship recorded during tetani in papillary muscles. Protein levels of troponin I (TnI) were measured in postischemic papillary muscles with the Western blot technique. Isometric contraction was depressed during the period of ischemia and remained low after 60 min of reoxygenation without a corresponding significant change of peak [Ca2+]i in the control group (n = 7). In contrast, the depression of isometric contraction was ameliorated during ischemia in muscle preparations in the presence of genistein (2 micro M; n = 8), and postischemic depressed myocardial contractility partially recovered after a 60-min reperfusion. The myofilament Ca2+ responsiveness was significantly increased in papillary muscles in the presence of genistein. Protein levels of TnI were reduced in postischemic papillary muscles, whereas genistein partially restored decreased protein levels of TnI. Our results reveal that genistein produces an effective attenuation of postischemic depressed myocardial function and improves myofibrillar Ca2+ responsiveness in rat myocardium.     

Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle

A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using alpha-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After alpha-toxin treatment the fibers developed tension as a function of Ca2+ concentration (EC50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5 X 10(-7) M Ca2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca2+ solution and a transient and maintained contraction in 5 X 10(-7) M Ca2+ solution. GTP-gamma-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca2+ and maintained tension revealed that NE and GTP-gamma-S cause increases in Ca2+ sensitivity of myofilament shifting the EC50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-gamma-S causes an increase in myofilament Ca2+ sensitivity and that G protein may be involved in receptor signal transduction system. alpha-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.     

Maximal Ca2+-activated force and myofilament Ca2+ sensitivity in intact mammalian hearts. Differential effects of inorganic phosphate and hydrogen ions

Myofilament Ca2+ sensitivity and maximal Ca2+-activated force are fundamental properties of the contractile proteins in the heart. Although these properties can be evaluated directly in skinned preparations, they have remained elusive in intact tissue. A novel approach is described that allows maximal Ca2+-activated force to be measured and myofilament Ca2+ sensitivity to be deduced from isovolumic pressure in intact perfused ferret hearts. Phosphorus nuclear magnetic resonance spectra are obtained sequentially to measure the intracellular inorganic phosphate (Pi) and hydrogen ion (H+) concentrations. After a period of perfusion with oxygenated, HEPES-buffered Tyrode solution, hypoxia is induced as a means of elevating [Pi]. The decline in twitch pressure can then be related to the measured increase in [Pi]. After recovery, hearts are perfused with ryanodine to enable tetanization and the measurement of maximal Ca2+-activated pressure. Hypoxia is induced once again, and maximal pressure is correlated with [Pi]. We then compare the relations between [Pi] and maximal pressure on the one hand, and [Pi] and twitch pressure on the other. If the two relations differ only by a constant scaling factor, then the decline in twitch pressure can be attributed solely to a decline in maximal pressure, with no change in myofilament sensitivity. We obtained such a result during hypoxia, which indicated that Pi accumulation decreases maximal force but does not change myofilament sensitivity. We compared these results with acidosis (induced by bubbling with 5% CO2). In contrast with Pi, the accumulation of H+ decreases twitch force primarily by shifting myofilament Ca2+ sensitivity. This approach in intact tissue has strengths and limitations complementary to those of skinned muscle experiments.     

Ca2+/calmodulin-dependent protein kinase II inhibition reduces myocardial fatty acid uptake and oxidation after myocardial infarction

An AMP-activated kinase (AMPK) signaling pathway is activated during myocardial ischemia and promotes cardiac fatty acid (FA) uptake and oxidation. Similarly, the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is also triggered by myocardial ischemia, but its function in FA metabolism remains unclear. Here, we explored the role of CaMKII in FA metabolism during myocardial ischemia by investigating the effects of cardiac CaMKII on AMPK-acetyl-CoA carboxylase (ACC), malonyl CoA decarboxylase (MCD), and FA translocase cluster of differentiation 36 (FAT/CD36), as well as cardiac FA uptake and oxidation. Moreover, we tested whether CaMKII and AMPK are binding partners. We demonstrated that diseased hearts from patients with terminal ischemic heart disease displayed increased phosphorylation of CaMKII, AMPK, and ACC and increased expression of MCD and FAT/CD36. AC3-I mice, which have a genetic myocardial inhibition of CaMKII, had reduced gene expression of cardiac AMPK. In post-MI (myocardial infarction) AC3-I hearts, AMPK-ACC phosphorylation, MCD and FAT/CD36 levels, cardiac FA uptake, and FA oxidation were significantly decreased. Notably, we demonstrated that CaMKII interacted with AMPK α1 and α2 subunits in the heart. Additionally, AC3-I mice displayed significantly less cardiac hypertrophy and apoptosis 2 weeks post-MI. Overall, these findings reveal a unique role for CaMKII inhibition in repressing FA metabolism by interacting with AMPK signaling pathways, which may represent a novel mechanism in ischemic heart disease.     

Alloxan reduces amplitude of ventricular myocyte shortening and intracellular Ca2+ without altering L-type Ca2+ current, sarcoplasmic reticulum Ca2+ content or myofilament sensitivity to Ca2+ in Wistar rats

Alloxan is widely used to induce diabetes mellitus in experimental animals. Recent studies have provided evidence that alloxan has direct actions on cardiac muscle contraction. The aim of this study was to further investigate the mechanisms underlying the effects of alloxan on ventricular myocyte shortening and intracellular Ca²⁺ transport. Amplitude of myocyte shortening was reduced in a dose-dependent manner as the concentration of alloxan was increased in the range 10^?7–10^?4 M. Amplitude of shortening was reduced (56.8 ± 6.6%, n = 27) by 10^?6 M alloxan and was partially reversed during a 10 min washout. Amplitude of the Ca²⁺ transient was also reduced (79.7 ± 2.9%, n = 29) by 10^?6 M alloxan. Caffeine-evoked sarcoplasmic reticulum Ca²⁺ release, fractional release of Ca²⁺, assessed by comparing the amplitude of electrically evoked with that of caffeine-evoked Ca²⁺ transients, and fura-2-cell length trajectory during the late stages of relaxation of myocyte twitch contraction were not significantly altered by alloxan. The amplitude of L-type Ca²⁺ current was not altered by alloxan. Alterations in sarcoplasmic reticulum Ca²⁺ transport, myofilament sensitivity to Ca²⁺, and L-type Ca²⁺ current do not appear to underlie the negative inotropic effects of alloxan.     

Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis in brain mitochondria

The differential scanning microcalorimetry and fluorescence methods, using probes ANS and pyrene, have been employed to study thermotropic behaviour of rat liver microsomes in the presence and absence of Mg2+. Addition of Mg2+ yields three partially reversible phase transitions at 18, 27 and 32 degrees C, respectively. A character of Mg2+-induced rearrangements in a membrane and their relation to a catalytic function of a cytochrome P-450-dependent enzymatic system is discussed.     

Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload

We have shown that intermittent interruption of immediate reflow at reperfusion (i.e., postconditioning) reduces infarct size in in vivo models after ischemia. Cardioprotection of postconditioning has been associated with attenuation of neutrophil-related events. However, it is unknown whether postconditioning before reoxygenation after hypoxia in cultured cardiomyocytes in the absence of neutrophils confers protection. This study tested the hypothesis that prevention of cardiomyocyte damage by hypoxic postconditioning (Postcon) is associated with a reduction in the generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h of hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned after the 3-h index hypoxia by three cycles of 5 min of reoxygenation and 5 min of rehypoxia applied before 6 h of reoxygenation. Relative to sham control and hypoxia alone, the generation of ROS (increased lucigenin-enhanced chemiluminescence, SOD-inhibitable cytochrome c reduction, and generation of hydrogen peroxide) was significantly augmented after immediate reoxygenation as was the production of malondialdehyde, a product of lipid peroxidation. Concomitant with these changes, intracellular and mitochondrial Ca(2+) concentrations, which were detected by fluorescent fluo-4 AM and X-rhod-1 AM staining, respectively, were elevated. Cell viability assessed by propidium iodide staining was decreased consistent with increased levels of lactate dehydrogenase after reoxygenation. Postcon treatment at the onset of reoxygenation reduced ROS generation and malondialdehyde concentration in media and attenuated cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase. Postcon treatment was associated with a decrease in intracellular and mitochondrial Ca(2+) concentrations. These data suggest that Postcon treatment reduces reoxygenation-induced injury in cardiomyocytes and is potentially mediated by attenuation of ROS generation, lipid peroxidation, and intracellular and mitochondrial Ca(2+) overload.     

Effects of MCC-135 on Ca2+ uptake by sarcoplasmic reticulum and myofilament sensitivity to Ca2+ in isolated ventricular muscles of rats with diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by delayed cardiac relaxation. Delayed relaxation is suggested to be associated with sarcoplasmic reticulum (SR) dysfunction and/or increase in myofilament sensitivity to Ca²⁺. Although MCC-135, an intracellular Ca²⁺-handling modulator, accelerates the delayed relaxation without inotropic effect in the ventricular muscle isolated from rats with diabetic cardiomyopathy, the underlying mechanism has not been fully understood. We tested the hypotheses that MCC-135 modulates Ca²⁺ uptake by SR and myofilament sensitivity to Ca²⁺. Wistar rats were made diabetic by a single injection of streptozotocin (40 mg/kg i.v.). Seven months later, the left ventricular papillary muscle was isolated and skinned fibers with and without functional SR were prepared by treatment of the papillary muscle with saponin to study SR Ca²⁺ uptake and myofilament sensitivity to Ca²⁺, respectively. In diabetic rats, SR Ca²⁺ uptake was decreased, which was related to decrease in protein level of SR Ca²⁺-ATPase determined by western blot analysis. MCC-135 enhanced SR Ca²⁺ uptake in diabetic rats, but not in normal rats. In diabetic rats, maximum force was decreased but force at diastolic level of Ca²⁺ was increased, without significant change in myofilament sensitivity to Ca²⁺ compared with normal rats. MCC-135 decreased force at any pCa tested (pCa 7.0-4.4), but had no significant effect on myofilament sensitivity to Ca²⁺ in diabetic rats. These results suggest that MCC-135 enhances SR Ca²⁺ uptake and shifts force-pCa curve downward without modulating myofilament sensitivity to Ca²⁺. These effects may contribute to positive lusitropic effect without inotropic effect of MCC-135 observed in the ventricular muscle of diabetic cardiomyopathy.     

Renal hypertension prevents run training modification of cardiomyocyte diastolic Ca2+ regulation in male rats.

The combined effects of endurance run training and renal hypertension on cytosolic Ca2+ concentration ([Ca2+]c) dynamics and Na+-dependent Ca2+ regulation in rat left ventricular cardiomyocytes were examined. Male Fischer 344 rats underwent stenosis of the left renal artery [hypertensive (Ht), n = 18] or a sham operation [normotensive (Nt), n = 20]. One-half of the rats from each group were treadmill trained for >16 wk. Cardiomyocyte fura 2 fluorescence ratio transients were recorded for 7 min during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degrees C. The rate of [Ca2+]c decline was not changed by run training in the Nt group but was reduced in the Ht group. At 7 min, cardiomyocytes were exposed to 10 mM caffeine in the absence of Na+ and Ca2+, which triggered sarcoplasmic reticular Ca2+ release and suppressed Ca2+ efflux via Na+/Ca2+ exchanger. External Na+ was then added, and Na+-dependent Ca2+ efflux rate was recorded. Treadmill training significantly enhanced Na+-dependent Ca2+ efflux rate under these conditions in the Nt group but not in the Ht group. These data provide evidence that renal hypertension prevents the normal run training-induced modifications in diastolic [Ca2+]c regulation mechanisms, including Na+/Ca2+ exchanger.     

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca(2+)](c)) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca(2+)) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca(2+) transients ([Ca(2+)](c)) and changes of mitochondrial Ca(2+) concentrations ([Ca(2+)](m)) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca(2+)](c) and [Ca(2+)](m) elevations elicited by K(+) pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca(2+)](m) was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca(2+)](c) transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca(2+) channel agonist Bay K 8644 enhanced K(+)-evoked [Ca(2+)](m) peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K(+) generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca(2+)](c) and [Ca(2+)](m) transients elicited by K(+), in PC12 cells overexpressing Bcl2, is related to the reduction of Ca(2+) entry through L-type Ca(2+) channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca(2+) channels, the subsequent Ca(2+) entry, and mitochondrial Ca(2+) overload.     

Phosphodiesterase inhibition and Ca2+ sensitization

Inhibitors of phosphodiesterase type III (PDE III) enhance cardiac contractile force by elevating the intracellular calcium concentration [Ca²⁺]_i by impairing cAMP degradation thus increasing cAMP levels. The drugs are more effective in healthy than in failing hearts since basal cAMP production is diminished in the latter. However, long term treatment with PDE-III inhibitors does not appear to be beneficial due to increased risk of potentially lethal arrhythmias caused by augmentation of [Ca²⁺]_i[1). This risk should be absent in Ca²⁺ sensitizers. Recently, thiadiazinone derivatives have been synthetized in which the potency for Ca²⁺ sensitization is many-fold larger than the potency for PDE-III inhibition. The Ca²⁺-sensitizing action resides in the [+]-enantiomers, while the [–]-enantiomers show weak PDE-III inhibition. In the enantiomer pair [+]-EMD 60263 and [–]-EMD 60264, only the former concentration-dependently increased force of contraction in isolated cardiac preparations and myocytes. In the Langendorff-perfused guinea-pig heart, force was reversibly increased, whereas [–]-EMD 60264 even produced a negative inotropic response despite of its PDE inhibitory activity. Heart rate, however, was reduced by both enantiomers. Perfusion pressure remained unaffected. The effects were fully reversible upon wash-out of the enantiomers. [+]-EMD 60263 also enhanced cell shortening of human myocytes from both normal and failing hearts. In contrast to the opposite effects on contractility, both enantiomers prolong the action potential duration by blocking the rapidly activating component of the delayed rectifier K⁺ current. Thus they also possess class III antiarrhythmic activity. The therapeutic potential of these agents has yet to be assessed in clinical studies.     

Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta

This study tested whether sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase regulates the ability of endothelin receptor antagonist to inhibit the endothelin-1 constriction. The endothelin A receptor antagonist BQ-123 (1 microM) completely relaxed constriction to 10 nM endothelin-1 in endothelium-denuded rat aorta. Challenge with cyclopiazonic acid (10 microM), a sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor, during the plateau of endothelin-1 constriction enhanced the constriction by approximately 30%. BQ-123 relaxed the endothelin-1 plus cyclopiazonic acid constriction by only approximately 10%. In contrast, prazosin (1 microM), an alpha-adrenergic receptor antagonist, still completely relaxed the 0.3 muM phenylephrine constriction in the presence of cyclopiazonic acid. Verapamil relaxed the endothelin-1 plus cyclopiazonic acid constriction by approximately 30%, whereas Ni(2+) and 2-aminoethoxydiphenyl borate, nonselective cation channel and store-operated channel blockers, respectively, completely relaxed the constriction. These results suggest that lowered sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase activity selectively decreases the ability of endothelin receptor antagonist to inhibit the endothelin A receptor. The decreased antagonism may be related to the opening of store-operated channels and subsequent greater internalization of endothelin A receptor.     

Expression of SERCA isoform with faster Ca2+ transport properties improves postischemic cardiac function and Ca2+ handling and decreases myocardial infarction

Myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, and myocyte death. Intracellular Ca(2+) overload with reduced activity of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a critical mechanism of this injury. Although upregulation of SERCA function is well documented to improve postischemic cardiac function, there are conflicting reports where pharmacological inhibition of SERCA improved postischemic function. SERCA2a is the primary cardiac isoform regulating intracellular Ca(2+) homeostasis; however, SERCA1a has been shown to substitute SERCA2a with faster Ca(2+) transport kinetics. Therefore, to further address this issue and to evaluate whether SERCA1a expression could improve postischemic cardiac function and myocardial salvage, in vitro and in vivo myocardial I/R studies were performed on SERCA1a transgenic (SERCA1a(+/+)) and nontransgenic (NTG) mice. Langendorff-perfused hearts were subjected to 30 min of global ischemia followed by reperfusion. Baseline preischemic coronary flow and left ventricular developed pressure were significantly greater in SERCA1a(+/+) mice compared with NTG mice. Independent of reperfusion-induced oxidative stress, SERCA1a(+/+) hearts demonstrated greatly improved postischemic (45 min) contractile recovery with less persistent arrhythmias compared with NTG hearts. Morphometry showed better-preserved myocardial structure with less infarction, and electron microscopy demonstrated better-preserved myofibrillar and mitochondrial ultrastructure in SERCA1a(+/+) hearts. Importantly, intraischemic Ca(2+) levels were significantly lower in SERCA1a(+/+) hearts. The cardioprotective effect of SERCA1a was also observed during in vivo regional I/R with reduced myocardial infarct size after 24 h of reperfusion. Thus SERCA1a(+/+) hearts were markedly protected against I/R injury, suggesting that expression of SERCA 1a isoform reduces postischemic Ca(2+) overload and thus provides potent myocardial protection.     

Upregulation of cardiac insulin-like growth factor-I receptor by ACE inhibition after myocardial infarction: potential role in remodeling.

This study evaluated the effects of angiotensin-converting enzyme (ACE) inhibition after myocardial infarction (MI) on cardiac remodeling and gene expression and localization of components (ligands, receptors, and binding proteins) of the cardiac insulin-like growth factor (IGF) system. After ligation of the coronary artery, rats were randomized to vehicle or ACE inhibitor (Captopril, 50 mg/kg/day) for 4 weeks. Blood pressure, cardiac remodeling, and components of the IGF system were localized in the heart using in situ hybridization (ISH) and immunohistochemistry (IHC). The average infarct size was 42%. There were regional differences in the expression of the IGF system after MI, with increased IGF-I mRNA abundance in the border (24-fold) and infarct (12-fold) and increased IGF-binding protein (IGFBP)-3 mRNA in all areas of the failing left ventricle (threefold). Captopril reduced blood pressure, attenuated cardiac remodeling, and caused a threefold increase in IGF-I receptor mRNA and protein in infarct, border zone, and viable myocardium (p<0.01). Captopril had no effect on IGF-I mRNA but further increased IGFBP-3 mRNA and protein in the border zone, (p<0.05). The changes in the cardiac IGF system following MI are highly localized, persist for at least 4 weeks, and can be modulated by ACE inhibition. These data suggest that the benefits of ACE inhibitors in attenuation of cardiac remodeling may be mediated in part through increased expression of the IGF-I receptor sensitizing the myocardium to the positive effects of endogenous IGF-I.     

Influence of gender on the response to hemodynamic overload after myocardial infarction

After myocardial infarction (MI), the left ventricle (LV) undergoes ventricular remodeling characterized by progressive global dilation, infarct expansion, and compensatory hypertrophy of the noninfarcted myocardium. Little attention has been given to the response of remodeling myocardium to additional hemodynamic overload. Studies have indicated that gender may influence remodeling and the response to both MI and hemodynamic overload. We therefore determined 1) structural and function consequences of superimposing hemodynamic overload (systemic hypertension) on remodeling myocardium after a MI and 2) the potential influence of gender on this remodeling response. Male and female Dahl salt-sensitive and salt-resistant rats underwent coronary ligation, resulting in similar degrees of MI. One week post-MI, all rats were placed on a high-salt diet. Four groups were then studied 4 wk after initiation of high-salt feeding: MI female, MI female + hypertension, MI male, and MI male + hypertension. Hypertension-induced pressure overload resulted in additional comparable degrees of myocardial hypertrophy in both females and males. In females, hypertension post-MI resulted in concentric hypertrophy with no additional cavity dilation and no measurable scar thinning. In contrast, in males, hypertension post-MI resulted in eccentric hypertrophy, further LV cavity dilation, and scar thinning. Physiologically, concentric hypertrophy in post-MI hypertensive females resulted in elevated contractile function, whereas eccentrically hypertrophied males had no such increase. Female gender influences favorably the remodeling and physiological response to hemodynamic overload after large MI.     

Pirenzepine prevents diethyl pyrocarbonate inhibition of central CO2 sensitivity

Application by pledget of the M1-antimuscarinic receptor agent pirenzepine (40 mM) to the rostral chemosensitive areas of the ventrolateral medulla in anesthetized, paralyzed, vagotomized, glomectomized, and servoventilated cats inhibited the slope of the integrated phrenic response to CO2 by 32.5% (P less than 0.03) and the maximum value by 21.1% (P less than 0.01). Similar application of the imidazole-histidine blocking agent diethyl pyrocarbonate (DEPC) decreased the slope by 40.3% (P less than 0.01) and the maximum value by 29.3% (P less than 0.05). Both responses confirm previous results. DEPC treatment decreased the effectiveness of subsequent pirenzepine application such that although slope and maximum were further decreased, the values were not significantly different from those after DEPC. Pirenzepine treatment prevented any subsequent DEPC inhibitory effect. The results raise the possibility that the inhibitory effects of DEPC on CO2 chemosensitivity are via muscarinic receptors and that muscarinic receptor involvement in CO2 chemosensitivity requires the presence of imidazole-histidine. Analysis by scintillation counting of successive 100-micron sections of medulla after rostral area application of [3H]pirenzepine indicated that the pirenzepine and DEPC effects are most probably within 2.0 mm of the ventral surface as measured from the midline, well away from the dorsal and ventral respiratory group neurons.     

Ca2+-calmodulin-dependent phosphorylation and passive transport of Ca2+ in the myocardial sarcolemma

Highly purified vesicles of rabbit myocardium sarcolemma with predominant inside-out orientation possess the Ca2+-calmodulin-dependent protein kinase activity. At optimal concentrations of calmodulin (0.5 microM) and Ca2+ (0.1 mM), the activity of protein kinase is 0.21 nmol 32P X min X mg of protein. The Km(app) value for ATP is 3.0 X 10(-6) M, V = 0.27 nmol 32P X mg of protein X min. Endogenous Ca2+-calmodulin-dependent protein kinase phosphorylates four protein substrates in sarcolemmal vesicles (Mr = 145, 22, 11.5, and 6-8 KD). Studies with passive efflux of Ca2+ from the SL vesicles showed that the Ca2+-calmodulin-dependent phosphorylation of protein components of sarcolemma inhibits this reaction.