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H C Hartzell

Publications and source records attributed to H C Hartzell.

At least 55 records · Page 3Linked to original sources
PubMed1990

Regulation of calcium current by low-Km cyclic AMP phosphodiesterases in cardiac cells.

The voltage-gated Ca2+ current (ICa) in cardiac myocytes is regulated by cAMP-dependent phosphorylation. Although the regulation of ICa via mechanisms involving modulation of cAMP synthesis is well understood, the regulation of cAMP degradation has been less thoroughly investigated. The goal of the present study was to investigate the participation of different subclasses of cAMP phosphodiesterase (PDE) in regulating cAMP-dependent phosphorylation of Ca2+ channels in frog ventricular myocytes. Cardiomyocytes were isolated enzymatically and mechanically and were patch-clamped using the whole-cell configuration of the patch-clamp technique. The effects of various low-Km cAMP PDE inhibitors on ICa were examined. None of the inhibitors tested [milrinone, indolidan, 1-methyl 3-isobutyl xanthine (MIX), rolipram, or Ro 20-1724] were able to elevate ICa in the absence of elevated cAMP, although they all increased ICa in the presence of submaximal levels of cAMP. This result suggests that these compounds do not act directly on Ca2+ channels but rather modulate cAMP degradation. Half-maximal effects were observed with 1.4 microM milrinone and 3.4 microM MIX. Milrinone was effective when applied from either the extracellular or intracellular surface, whereas MIX was effective only when applied from the extracellular solution. In the presence of internal cGMP, which stimulates the cGMP-stimulated PDE, the low-Km cAMP PDE inhibitors had no effect on ICa, whereas high concentrations of MIX, which inhibit the cGMP-stimulated PDE, increased ICa. This would support the hypothesis that cGMP-stimulated PDE either has a much stronger capacity to hydrolyze cAMP or is more efficiently coupled to Ca2+ channels than the low-Km cAMP PDEs.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes.

The effects of changes in intracellular and extracellular free ionized [Mg2+] on inactivation of ICa and IBa in isolated ventricular myocytes of the frog were investigated using the whole-cell configuration of the patch-clamp technique. Intracellular [Mg2+] was varied by internal perfusion with solutions having different calculated free [Mg2+]. Increasing [Mg2+]i from 0.3 mM to 3.0 mM caused a 16% reduction in peak ICa amplitude and a 36% reduction in peak IBa amplitude, shifted the current-voltage relationship and the inactivation curve approximately 10 mV to the left, decreased relief from inactivation, and caused a dramatic increase in the rate of inactivation of IBa. The shifts in the current-voltage and inactivation curves were attributed to screening of internal surface charge by Mg2+. The increased rate of inactivation of IBa was due to an increase in both the steady-state level of inactivation as well as an increase in the rate of inactivation, as measured by two-pulse inactivation protocols. Increasing external [Mg2+] decreased IBa amplitude and shifted the current-voltage and inactivation curves to the right, but, in contrast to the effect of internal Mg2+, had little effect on the inactivation kinetics or the steady-state inactivation of IBa at potentials positive to 0 mV. These observations suggest that the Ca channel can be blocked quite rapidly by external Mg2+, whereas the block by [Mg2+]i is time and voltage dependent. We propose that inactivation of Ca channels can occur by both calcium-dependent and purely voltage-dependent mechanisms, and that a component of voltage-dependent inactivation can be modulated by changes in cytoplasmic Mg2+.

Animals↗
PubMed1989

Modulation of the delayed rectifier potassium current in frog cardiomyocytes by beta-adrenergic agonists and magnesium.

1. The regulation of IK and ICa were studied in single cells isolated from bull-frog atrium using the whole-cell configuration of the patch clamp and a perfused patch pipette. 2. IK was increased approximately 50-100% and ICa was increased approximately 6-10 times by 1 microM-isoprenaline, 5 microM-forskolin, or internal perfusion with 30 microM-cyclic AMP. The effects of cyclic AMP and isoprenaline were not additive. The shape of the concentration-response curves and the EC50 values for the effects of cyclic AMP on ICa and on IK were very similar (2.3 microM for IK and 1.7 microM for ICa). 3. Elevation of intracellular cyclic AMP had a similar effect on IK regardless of whether ICa was blocked with Cd2+ or not. Increasing ICa with dihydropyridine Ca2+ channel agonists had no effect on IK amplitude. 4. Isoprenaline or cyclic AMP caused an increase in the fully-activated IK and also shifted the activation curves to more negative potentials in most cells. The shift in the activation curve was reversible and was also observed when ICa was blocked with Cd2+. The rate of activation of IK was increased and the rate of deactivation of IK was slowed by isoprenaline. 5. After breaking the membrane patch and initiating whole-cell recording, IK ran down with time in about 50% of the cells examined when the intracellular solution contained 1 mM [Mg2+]. In contrast, when the solution contained 0.3 mM [Mg2+], rundown was almost never observed. Internal perfusion with increasing concentrations of [Mg2+] caused reversible decreases in the maximum amplitude of IK and shifted the IK activation curve slightly to more negative potentials, but had negligible effects upon the shape or the curvature of the fully activated current-voltage relationship.

Action Potentials↗
PubMed1988

Effects of intracellular free magnesium on calcium current in isolated cardiac myocytes.

Magnesium ions play a fundamental role in cellular function, but the effects of changes in the concentration of intracellular ionized magnesium ([Mg2+]i) on cell physiology have only recently received experimental attention. Increasing [Mg2+]i from 0.3 to 3.0 mM in cardiac cells by internal perfusion has only small effects on the basal voltage-gated calcium current (ICa) or on ICa elevated by dihydropyridine calcium channel agonists. In contrast, ICa elevated by cyclic adenosine monophosphate (cAMP)-dependent phosphorylation decreases by more than 50 percent. The effect of [Mg2+]i is not due to changes in the concentration of cAMP or in the velocity of phosphorylation but rather appears to be a direct effect on the phosphorylated channel or on channel dephosphorylation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Inactivation, reactivation and pacing dependence of calcium current in frog cardiocytes: correlation with current density.

1. Ca2+ currents were measured in single cells isolated from frog ventricle using the whole-cell patch clamp technique and a perfused pipette. K+ currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs+. 2. A single type of Ca2+ current (ICa) was found in these cells. The current activated at voltages positive to -30 mV, exhibited a symmetrical current-voltage relationship with a peak at 0 mV, and was slowly inactivating with Ba2+ as charge carrier. 3. Large variations in ICa amplitude were observed from cell to cell (ICa at 0 mV = 293.1 +/- 283.3 pA; N = 152). These variations were not due simply to differences in cell membrane area, which was estimated by cell membrane capacitance (Cm), because the density of Ca2+ current (dICa = ICa/Cm) also varied significantly from cell to cell (1.3-28 pA/pF at 0 mV; mean +/- S.D. = 4.49 +/- 3.96; N = 152). 4. The inactivation curve of ICa was a complex function of membrane potential. 200 ms pre-pulses to voltages between -60 and +20 mV progressively inactivated ICa elicited by a subsequent test pulse with half-maximal inactivation occurring for pre-pulses to approximately -40 mV. With pre-pulses positive to +20 mV, ICa elicited by the test pulse became progressively larger. The degree of inactivation induced by a 200 ms depolarization to potentials more positive than +20 mV varied significantly from cell to cell, while no such variations were observed in the negative range of membrane potentials. 5. The time course of reactivation (i.e. removal from inactivation) of ICa at -80 mV often exhibited an overshoot. The amplitude of the overshoot varied between 100% (i.e. no overshoot) and approximately 180% in eighty-one cells. 6. The degree of inactivation at positive potentials (+100 mV) and the amplitude of the overshoot were strongly correlated with the Ca2+ current density. The overshoot was more pronounced, the reactivation was faster, and the inactivation at positive potentials was less in cells with lower ICa density. 7. Increasing the stimulation frequency from 0.125 to 2 Hz induced a positive staircase of ICa in cells with ICa density less than 2 pA/pF and a negative staircase in cells with ICa density greater than 3 pA/pF. 8. Perfusing the patch pipette with 5 mM-BAPTA instead of EGTA reduced the amplitude of the overshoot and slightly slowed the inactivation kinetics. Replacing extracellular Ca2+ ions by Ba2+ ions completely suppressed the overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1988

Role of phosphodiesterase in regulation of calcium current in isolated cardiac myocytes.

It has previously been shown that intracellular perfusion of isolated cardiac myocytes with cGMP reduces the amplitude of the trans-sarcolemmal calcium current (ICa) elevated by cAMP-dependent mechanisms. To test the hypothesis that cGMP acts by stimulating a cyclic nucleotide phosphodiesterase (PDE) activity, PDE activity and the effects of methylisobutylxanthine (MIX), a PDE inhibitor, on ICa were examined in cardiomyocytes dissociated from frog ventricle. PDE activity was determined by measuring hydrolysis of [33P]cAMP in subcellular fractions. Using 100 microM cAMP as substrate, approximately 50% of the PDE activity was found in the 20,000 x g particulate fraction. Basal activity in this fraction had a Vmax of 15.4 nmol [corrected] of cAMP hydrolyzed/min/mg of protein and a Km of 113 microM cAMP. The PDE activity of the particulate fraction was stimulated significantly by cGMP. Half-maximal stimulation was observed with 1.1 microM cGMP. This value is virtually identical to the value for the concentration of intracellular cGMP that produced a half-maximal reduction of cAMP-elevated ICa in electrophysiological experiments. The cGMP-stimulated PDE activity had a Vmax of 9.5 nmol/min/mg [corrected] and a Km of 12.3 microM cAMP. MIX (100 microM) selectively inhibited the cGMP-stimulated PDE activity (IC50 = 20 microM). To determine whether PDEs modulate the amplitude of ICa, the effects of MIX were examined on basal ICa and cAMP-elevated ICa. MIX produced small increases in the basal ICa and increased ICa in the presence of 1 microM intracellular cAMP. MIX at 100 microM potentiated the effects of submaximal doses of isoproterenol and shifted the dose-response curve for cAMP to the left but did not affect the dose-response curve for 8-bromo-cAMP. MIX reversed the effect of cGMP on the cAMP-elevated ICa. We conclude that cyclic nucleotide PDEs play an important role in modulating the cardiac calcium current. The hypothesis that cGMP inhibits the cAMP-elevated ICa by activating a PDE is supported by the finding that MIX inhibited both the cGMP-stimulated PDE activity and the effect of cGMP on ICa at similar concentrations.

1-Methyl-3-isobutylxanthine↗
PubMed1987

A quantitative analysis of the acetylcholine-activated potassium current in single cells from frog atrium.

The K+ current activated by acetylcholine (ACh) in single cells from the frog atrium was analyzed using the whole-cell patch clamp technique. The ACh current was analyzed quantitatively by subtracting the currents elicited in response to voltage steps in the presence and absence of a steady bath-application of 1 microM ACh. The net ACh currents were voltage- and time-dependent. With depolarizing jumps, the ACh-activated current declined from an instantaneous peak to a new steady level. With hyperpolarizations, the instantaneous current change was followed by a time-dependent increase in current. The current relaxations were well fitted by the sum of two exponentials with time constants of approximately 20 ms and approximately 300 ms at -120 mV. Both time constants decreased with depolarization. The current-voltage relationship inwardly-rectified. This inward rectification was due both to a decrease in the single channel conductance and a decrease in the number of open channels with depolarization. The ACh-activated K+ current differs from the background K+ current in several respects. The kinetics of the background K+ current are much more rapid and the background K+ channel passes much less current in the outward direction than the ACh channel.

Acetylcholine↗
PubMed1987

Cyclic guanosine 3',5'-monophosphate regulates the calcium current in single cells from frog ventricle.

1. The effect of intracellular perfusion with cyclic AMP and cyclic GMP on Ca2+ current (ICa) was studied in single cells isolated from frog ventricle using the whole-cell patch-clamp technique and a perfused pipette. 2. Intracellular perfusion with cyclic GMP (0.1-20 microM) had no effect on the basal ICa. However, when ICa was increased by isoprenaline or by intracellular perfusion with cyclic AMP, perfusion with cyclic GMP (20 microM) reduced ICa by an average of 67%. The effect of cyclic GMP on ICa elevated by cyclic AMP was reversible. A half-maximal effect of cyclic GMP was observed at 0.6 microM. Cyclic GMP had no significant effect on the shape of the ICa current-voltage relationship. 3. The effect of cyclic GMP was specific to the 3',5' form; 2',3'-cyclic GMP had no effect. 4. The effect of cyclic GMP was apparently not mediated by stimulation of cyclic-GMP-dependent protein kinase because 8-bromo-cyclic GMP, a very potent activator of the protein kinase, was without effect. 5. Cyclic GMP had no effect on ICa elevated by the non-hydrolysable 8-bromo-cyclic AMP. The effect of cyclic GMP on cyclic-AMP-elevated ICa was partially blocked by the phosphodiesterase inhibitor, methylisobutylxanthine. Thus, it was hypothesized that the effect of cyclic GMP was mediated by hydrolysis of cyclic AMP as a result of a stimulation of a cyclic nucleotide phosphodiesterase by cyclic GMP. 6. The dose-response curve for cyclic AMP on ICa was well fitted by the Michaelis equation with a K50 (i.e. concentration of cyclic AMP at which response is 50% of the maximum) of 0.7 microM and a maximal 11-fold stimulation of ICa. Cyclic GMP shifted the curve one log unit to the right and decreased the maximal stimulation to 8.6-fold. Thus, the effect of cyclic GMP appeared uncompetitive. 7. The products of cyclic AMP and cyclic GMP hydrolysis, 5'-AMP and 5'-GMP, had no effect on ICa. Furthermore, strong buffering of intracellular pH did not reduce the effect of cyclic GMP. 8. It is proposed that cyclic-GMP-stimulation of a cyclic nucleotide phosphodiesterase may be one of several mechanisms by which acetylcholine regulates ICa.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Comparison of effects of acetylcholine on calcium and potassium currents in frog atrium and ventricle.

1. Ca2+ and K+ currents were measured in single atrial and ventricular myocytes from frog heart with the whole-cell patch-clamp technique. 2. K+ currents were blocked with intra- and extracellular Cs+ and the fast Na+ current was blocked with tetrodotoxin (TTX). The Ca2+ current (ICa) was evoked by a depolarizing pulse from -80 to 0 mV. ICa was larger in ventricular (3.4 +/- 2.5 microA/cm2) than atrial (1.6 +/- 2.5 microA/cm2) myocytes. 3. Acetylcholine (ACh) had no effect on basal ICa when K+ currents were blocked with Cs+ or Ba2+. Isoprenaline increased ICa and ACh reduced the isoprenaline-stimulated current to basal levels. 4. In contrast, when K+ currents were not blocked, ACh reduced the net inward current and increased the outward current at the end of the depolarizing pulse. The outward current was studied in the presence of Cd2+ to block ICa. The steady-state current-voltage relationship inwardly rectified and reversed near the K+ reversal potential (EK). The magnitude of the steady-state ACh-activated K+ current at 0 mV was 1.0 +/- 0.7 microA/cm2 in ventricular cells and 3.67 +/- 1.7 microA/cm2 in atrial cells. 5. With depolarization, the outward current increased instantaneously and then decreased to a new steady level. The first phase of the decay occurred with a time constant similar to that of the activation of ICa. The Cd2+-sensitive current (corresponding to ICa) was obtained by subtracting currents in the presence and absence of Cd2+. The Cd2+-sensitive current was not affected by ACh. 6. The apparent effect of ACh on basal ICa can be explained quantitatively by activation of a time-dependent K+ current by ACh that contaminates ICa.

Acetylcholine↗
PubMed1987

Effect of forskolin and acetylcholine on calcium current in single isolated cardiac myocytes.

The effect of extracellular and intracellular application of forskolin on the voltage-sensitive calcium current, ICa, was studied in myocytes isolated from frog ventricle. Myocytes were isolated by enzymatic dissociation, and ICa was measured using the whole-cell configuration of the patch clamp technique modified to permit intracellular perfusion of various substances. Intracellular perfusion with forskolin (0.1 to 10 microM) had a negligible effect on ICa: ICa was increased 15 +/- 13% (mean +/- SE; N = 5). In contrast, superfusion of the cell with forskolin increased ICa significantly. The EC50 for the forskolin effect was 0.4 microM. A maximal 4.5-fold increase in ICa occurred with 3 microM forskolin. This is somewhat less than the maximal response to isoprenaline seen in this same series of experiments. The effects of forskolin, isoprenaline, and intracellular cAMP were not additive. In contrast, the effects of isoprenaline or intracellular cAMP and calcium channel agonists, such as Sandoz (+)202-791, were additive. This supports the hypothesis that the positive inotropic effects of forskolin are at least partly mediated by an increase in intracellular cAMP and a stimulation of ICa. The effects of forskolin were antagonized by acetylcholine (1 microM) or intracellular perfusion with cGMP. Acetylcholine on the average decreased forskolin-stimulated ICa 57 +/- 11% (N = 17). The relevance of these results to the suggestion that acetylcholine acts by mechanisms other than inhibition of adenylate cyclase is discussed.

Acetylcholine↗
PubMed1986

A time-dependent and voltage-sensitive K+ current in single cells from frog atrium.

A quantitative description of the time-dependent and voltage-sensitive outward currents in heart has been hampered by the complications inherent to the multicellular preparations previously used. We have used the whole-cell patch-clamp technique to record the delayed outward K+ current, IK, in single cells dissociated from frog atrium. Na+ currents were blocked with tetrodotoxin and Ca2+ currents with Mn2+ or Cd2+. After depolarizations from -50 mV to potentials positive to -30 mV, a time-dependent outward current was observed. This current has been characterized according to its steady state activation, kinetics, and ion transfer function. The current is well described as a single Hodgkin-Huxley conductance. The deactivation of the current is a single exponential. Activation of the current is sigmoid and is fitted well by raising the activation variable to the second power. The reversal potential of IK is near EK and shifts by 57 mV/10-fold change in [K+]o. This suggests that the current is carried selectively by K ions. The threshold for activation is near -30 mV. IK is maximally activated positive to +20 mV and shows no inactivation. The fully activated current-voltage relationship is linear between -110 and +50 mV. Neither Ba2+ (250 microM) nor Cd2+ (100 microM) affects IK.

Action Potentials↗
PubMed1986

Mechanism of action of acetylcholine on calcium current in single cells from frog ventricle.

Ca currents (ICa) were measured by whole-cell patch clamp in single cells isolated from frog ventricle in which K currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs. Inward currents elicited by depolarizing voltage steps from a holding potential of -80 mV were blocked completely by 0.5 mM-Cd. The quality of the voltage clamp was assessed using two patch electrodes on a single cell. One electrode was used in the voltage-clamp mode to measure membrane currents and the other in current-clamp to measure membrane potential. Ca currents as large as 2 nA were well clamped in cells as long as 210 micron. Acetylcholine (ACh) had no effect on ICa in the absence of beta-adrenergic stimulation but reduced to control levels ICa elevated by isoprenaline. Nanomolar concentrations of ACh were able to reduce significantly ICa elevated by 2 microM-isoprenaline. ACh had no effect on the shape of the I-V curve, on the reactivation (recovery from inactivation), or on the inactivation of ICa. Although isoprenaline increased ICa by an average of 6.5-fold, it had no effect on the shape of the I-V curve or on the inactivation at test potentials negative to +40 mV. However, isoprenaline slowed the half-reactivation time from a control value of 120 +/- 10 ms (mean +/- S.D.) to 153 +/- 12 ms at -80 mV. The effect of cyclic AMP on ICa was investigated using two patch electrodes, one filled with cyclic AMP. Maximal effects of cyclic AMP were observed with 5 microM-cyclic AMP in the pipette. Maximal ICa was recorded several minutes after breaking the patch with the second electrode. After removing the cyclic-AMP-containing electrode, ICa declined to control levels after approximately 10 min. 5 microM-cyclic AMP in the patch electrode increased ICa by an average of 6.9-fold, but had no effect on the shape of the I-V curve or on inactivation. Cyclic AMP had a slowing effect on reactivation (half-reactivation time = 155 +/- 24 ms) similar to that of isoprenaline. ACh (1-10 microM) did not reduce ICa elevated with cyclic AMP (0.1-20 microM-cyclic AMP in the pipette). With low concentrations of cyclic AMP in the pipette (0.1 microM), isoprenaline augmented ICa, but with 5 microM-cyclic AMP in the pipette, isoprenaline was incapable of increasing ICa further. These results suggest that the decrease of ICa produced by ACh can be explained solely by decreases in cyclic AMP levels.

Acetylcholine↗
PubMed1985

Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase.

C-protein, a component of the thick filaments of striated muscles, is reversibly phosphorylated and dephosphorylated in heart. It has been hypothesized that C-protein may be involved in regulating contraction, because the extent of C-protein phosphorylation correlates with the rate of cardiac relaxation. To test this hypothesis, the effects of phosphorylated and unphosphorylated C-protein on the actin-activated ATPase activity of myosin filaments prepared from DEAE-Sephadex-purified myosin were examined. Unphosphorylated C-protein (0.1 microM to 1.5 microM) stimulated actin-activated myosin ATPase activity in a dose-dependent manner. With a myosin: C-protein molar ratio of approximately 1, actin-activated myosin ATPase activity was elevated up to 3.2 times that of the control. Phosphorylated C-protein (2.5 mol PO4/mol C-protein) stimulated the activity somewhat less (2.5 times that of control). The stimulation of ATPase activity by C-protein was due to an increase in the Vmax value (from 0.25/second to 0.62/second) and a decrease in the Km value (from 11.9 microM to 6.7 microM). The addition of C-protein to actomyosin solutions produced an increase in the light-scattering of the actomyosin solution and a distinct precipitation of the actomyosin with time. Phosphorylated C-protein had a smaller effect on light-scattering than dephosphorylated C-protein. C-protein had a negligible effect on Ca-ATPase, EDTA-K-ATPase, or Mg-ATPase activities in the absence of actin. C-protein had only small effects on the actin-activated ATPase of heavy meromyosin. These results suggest that C-protein stimulates actin-activated myosin ATPase activity by enhancing the formation of stable aggregates between actin and myosin filaments.

Actin Cytoskeleton↗
PubMed1985

Structure of C protein purified from cardiac muscle.

C protein is a component of the thick filament of striated muscles. Although the function of C protein remains unknown, a variety of evidence suggests that C protein may regulate actin-myosin interaction or be involved in structural support or elasticity of the sarcomere. We have previously proposed (Hartzell, H. C., 1984, J. Gen. Physiol., 83:563-588) that C protein is involved in regulating twitch relaxation in cardiac muscle. To gain further insight into the function of C protein, we have studied the structure of C protein purified from chicken heart. C protein was purified from extracts of detergent-washed myofibrils by sequential hydroxylapatite and DEAE-Sephacel chromatography. C protein was judged greater than 95% pure by SDS PAGE. The polypeptide subunit had a molecular weight of 155,000 and the native molecule sedimented on linear sucrose or glycerol gradients at 4-5S. For electron microscopy, purified C protein was dialyzed and diluted into a volatile buffer in 50% glycerol, aspirated onto mica, dried under vacuum, and rotary platinum-shadowed. Replicas revealed particles of relatively homogeneous overall dimensions. Over half of the particles were V-shaped. The "arm" lengths of the V-shaped particles were 22 +/- 4.5 nm (SD). Gel filtration on Sephacryl S-300 demonstrated that purified C protein had a Stokes' radius of 5.07 nm. Measurements of viscosity gave an intrinsic viscosity of 16.5 cm3/g. These data are consistent with the electron microscopic data and suggest that C protein in heart muscle is asymmetric. The C protein molecule is large enough to extend from the surface of a thick filament to adjacent thin or thick filaments.

Animals↗
PubMed1985

Reduction of muscarinic acetylcholine receptor number and affinity by an endogenous substance.

We examined the soluble fraction from homogenates of 12-day embryonic chick heart for the presence of an endogenous modulator of muscarinic acetylcholine receptors (mAChR). Homogenates were separated into 100,000 g soluble and crude membrane fractions by differential centrifugation. Aliquots of membranes were incubated in the presence or absence of the soluble fraction and the muscarinic antagonist, [3H]quinuclidinyl benzilate ( [3H]QNB), and the data subjected to Scatchard analysis. In the presence of the soluble fraction, mAChR number decreased up to 70% and the affinity for [3H]QNB decreased six- to eightfold. These results suggested that an endogenous soluble factor (ESF) affected cholinergic ligand binding to the receptor. The amount of ESF extracted from less than 10 mg of brain was sufficient to reduce by 50% [3H]QNB binding to 50 fmol mAChR. ESF activity was partially purified by heat and acid treatment. The loss of receptors was dependent upon the amount of ESF added and was time dependent. QNB protected some receptors from loss due to ESF. The change in mAChR affinity for [3H]QNB was observed only if ESF was present continuously during the [3H]QNB binding assay. Ultrafiltration and gel filtration showed that ESF was less than 10,000 daltons and probably less than 700 daltons. ESF activity was blocked by EDTA. However, ESF was not a divalent cation since it was base labile, and removal of divalent cations with Chelex-100 did not inhibit ESF activity. ESF activity was also blocked by catechol, catecholamines, ascorbate, and dithiothreitol. ESF was present in embryonic but not in adult heart.

Animals↗
PubMed1984

Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases.

C-protein, a component of the thick filament of striated muscles, becomes phosphorylated in response to beta-adrenergic receptor stimulation and dephosphorylated in response to cholinergic receptor stimulation in heart. We have purified C-protein in high yield from cardiac muscle (approximately 50% yield: 0.3 mg of C-protein/g of frozen chicken heart). C-protein has a molecular weight on sodium dodecyl sulfate polyacrylamide gels of 155,000 but the native protein migrates as a globular protein of 209,000 daltons in gel filtration on Sephacryl S-300, suggesting that it is an asymmetric molecule composed of a single 155,000-dalton polypeptide. C-protein from chicken cardiac muscle has an amino acid composition similar to that of C-proteins from other muscles. The purified protein contains approximately 0.2 mol of phosphate/mol of C-protein. The purified C-protein has no endogenous protein phosphatase activity but does exhibit protein kinase activity in the presence of calcium and calmodulin (approximately 160 pmol of phosphate incorporated/min/mg of C-protein). This endogenous kinase catalyzes the incorporation of approximately 1 mol of phosphate/mol of C-protein. C-protein is an excellent substrate for catalytic subunit of cAMP-dependent protein kinase (Km = 4 microM, Vmax = 18.6 mumol/min/mg). Phosphorylation by catalytic subunit of cAMP-dependent protein kinase exhibits a broad pH optimum between pH 8 and 9 and results in the incorporation of up to 3 mol of phosphate/mol of C-protein. Phosphate is incorporated into 3-5 different sites at both phosphothreonine and phosphoserine residues. The phosphorylated C-protein does not differ from unphosphorylated C-protein with regard to Stokes radius, migration on sodium dodecyl sulfate-polyacrylamide gels, or UV spectrum.

Animals↗