Rat Cardiac Ventricular Myocytes Research Articles

Calcium‐induced release of strontium ions from the sarcoplasmic reticulum of rat cardiac ventricular myocytes

1. The effects of strontium ions, Sr2+, on Ca(2+)-dependent feedback mechanisms during excitation-contraction coupling were examined in voltage-clamped rat ventricular myocytes in which intracellular [Ca2+] and [Sr2+] were monitored with the fluorescent indicator, indo-1. 2. Voltage clamp depolarizations and caffeine applications during superfusion in Ca(2+)-free, Sr(2+)-containing solutions were employed to exchange intracellular Ca2+ with Sr2+. Myocytes were loaded with Sr2+ by applying voltage clamp depolarizations during superfusion in Na(+)-free, Sr(2+)-containing solutions. 3. Caffeine applications produced large fluorescence transients in Sr(2+)-loaded cells. Thus, Sr2+ could be sequestered and released from the sarcoplasmic reticulum. 4. Ca2+ influx, but not Sr2+ influx, via sarcolemmal Ca2+ channels evoked ryanodine-sensitive fluorescence transients in Sr(2+)-loaded cells. These results demonstrated that Ca2+ influx-induced Sr2+ release (CISR) from the sarcoplasmic reticulum occurred in these experiments, even though Sr2+ influx-induced Sr2+ release was not observed. 5. The amplitude of the Ca2+ influx-induced fluorescence transient was 17 +/- 1% of the caffeine-induced transient (n = 5 cells), an indication that fractional utilization of Sr2+ sequestered in the sarcoplasmic reticulum during CISR was low. 6. With increased Sr2+ loading, the amplitude of Ca2+ influx- and caffeine-induced fluorescence transients increased, but fractional utilization of sarcoplasmic reticulum divalent cation stores was independent of the degree of Sr2+ loading. These data suggest that Ca2+ influx directly activated the release of divalent cations from the sarcoplasmic reticulum, but mechanisms promoting positive feedback of Sr2+ release were minimal during CISR. 7. By comparison, in Ca(2+)-loaded myocytes, Ca2+ influx-induced Ca2+ release (CICR) utilized a greater fraction of caffeine-releasable stores than CISR. Fractional utilization of Ca2+ stores during CICR increased with the degree of Ca2+ loading. 8. Taken together, these results suggest that Ca(2+)-dependent feedback mechanisms play a major role in determining the extent of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling under a wide range of conditions.

Electrogenic K+ transport by the Na(+)-K+ pump in rat cardiac ventricular myocytes.

1. The involvement of electrogenic reaction steps in K+ transport by the Na+, K(+)-ATPase was determined in rat cardiac ventricular myocytes using whole-cell patch clamp techniques. 2. Under K(+)-K+ exchange conditions and in the presence of extracellular K+ or Tl+ at concentrations that stimulated submaximal levels of steady-state Na+,K(+)-ATPase activity, ouabain-sensitive transient currents were observed during ('on') and after ('off') step changes in membrane potential (Vm). 3. The quantity of charge moved during the transient currents depended, in a saturable manner, on the magnitude of the voltage step. Maximal ouabain-sensitive 'on' and 'off' charges were calculated to be 9.6 +/- 0.9 and 9.1 +/- 0.4 fC pF-1 (n = 4), respectively, with an effective valeney of 0.48 +/- 0.07 (n = 7). 4. Kinetics of the transient currents were independent of Vm and Tl+o at positive potentials, but became more rapid at increasingly negative Vm values in an ion concentration-dependent fashion. 5. Those data demonstrate that electrogenic steps participate in K+ transport by the Na+,K(+)-ATPase and that the electrogenic step is extracellular ion binding. 6. The temperature- and Vm-dependent properties of transient charge movements were compared under K(+) -K+ and Na(+) -Na+ exchange conditions. The data suggest that extracellular K+ and Na+ binding occur at different sites in the enzyme or to different enzyme conformations. The sum of the effective valencies, 1.14 +/- 0.12, demonstrates that the electrogenicity of extra-cellular ion binding can explain the Vm dependence of ion transport by the Na+,K(+)-ATPase.

Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells

The Na/K pump current I p of single HEK293 cells either untransfected (endogenous I p) or transfected with the α1 subunit of the rat Na/K pump (exogenous I p) was investigated in Na-containing solution by means of whole-cell recording at 30°C. The endogenous I p was irreversibly blocked by 10 −4 M ouabain or 2·10 −4 M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF −1 at 0 mV and 5.4 mM K o. It was half maximally activated at 1.5 mM K o and increased linearly with depolarization over the entire voltage range studied (−80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant α1 subunit of the rat Na/K pump showed an I p in the presence of 10 −4 M ouabain and 2·10 −4 M DHO, respectively. This exogenous I p was reversibly blocked by 10 −2 M ouabain. Half maximal activation of the exogenous I p occurred at 1.7 mM K o. Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I p of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I p in HEK293 cells is generated by the α1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.

Characterization of calcium-dependent forms of protein kinase C in adult rat ventricular myocytes.

The presence and subcellular localization of the Ca2+-dependent protein kinase C (PKC) isoforms alpha and beta were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was measured in cytosolic and particulate fractions prepared from control myocytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-oleoyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studies detected no Ca2+-/phospholipid-dependent histone kinase activity in either subcellular fraction. To reproducibly observe Ca2+-/phospholipid-dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-dependent protein kinase were essential. MonoQ chromatography of cytosolic and particulate fractions resulted in three peaks of Ca2+/phospholipid-dependent protein kinase activity. In the cytosolic fraction a large peak of activity eluted at 230-300 mM NaCl. Isoform-specific antisera indicated both PKC alpha and PKC beta were present. In the particulate fraction two peaks of Ca2+-/phospholipid-dependent protein kinase activity, both containing PKCa immunoreactivity, were observed. The larger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at lower salt concentrations contained a Ca2+-/phospholipid-independent histone kinase activity. This peak of kinase activity contained PKC alpha immunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenzyme of PKC alpha whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, the peak of kinase activity eluting at 230-300 mM NaCl contained PKC alpha in the form of an 80-kDa doublet; this suggested the presence of autophosphorylated PKC. Incubation of the myocytes with PMA, but not OAG, resulted in translocation of PKC from the cytosolic to the particulate fraction. Curiously, a transient decrease in PKC activity was observed in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated adult rat cardiac ventricular myocytes contain both PKC alpha and PKC beta, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG.

Characterization of a stimulatory adenosine A2a receptor in adult rat ventricular myocyte.

The expression and function of stimulatory adenosine A2 receptor on the cardiac myocyte is not well defined. The objective of the present study is to characterize the A2a receptor in adult rat cardiac ventricular myocytes. After selection of an optimal lot of collagenase for myocyte isolation and for consistent measurement of adenosine-mediated responses, the A1-receptor pathway was inactivated by pertussis toxin and by the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine. Effects of the adenosine agonist and antagonist or cardiac myocyte contractile amplitude and on adenosine 3',5'-cyclic monophosphate (cAMP) levels were determined. The A2a-receptor-selective agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoade nos ine (CGS-21680) caused a pronounced stimulation of myocyte contractile amplitude and an increase in the cAMP level, as did the nonselective agonists 5'-(N-ethylcarboxamido) adenosine (NECA) and adenosine. The A2a-receptor-selective antagonist 8-(3-chlorostyryl)caffeine blocked the NECA- and adenosine-induced positive inotropic response. Probing of myocyte RNA with a rat A2a-receptor cDNA demonstrated a 2.6-kb mRNA, corresponding to that encoding the A2a receptor. Together, data from contractile, cAMP, and RNA studies indicate that A2a receptors are expressed and are functionally coupled to stimulation of cAMP accumulation and cardiac contractility in adult rat ventricular myocytes.

Cocaine increases circulating levels of atrial natriuretic peptide and pro atrial natriuretic peptide N-terminal fragment in conscious rats

We examined the effects of intravenously administered cocaine (1 and 3 mg/kg) on haemodynamics (mean arterial pressure, heart rate and right atrial pressure), plasma immunoreactive atrial natriuretic peptide (immunoreactive ANP) and immunoreactive N-terminal peptide of proANP (immunoreactive N-terminal ANP) in conscious, chronically cannulated Sprague-Dawley rats. The direct effect of cocaine (10 −6–10 −4 M) was also studied in primary cultures of neonatal rat cardiac ventricular myocytes. Intravenous injection of 1 mg/kg or 3 mg/kg of cocaine caused an immediate peak rise in mean arterial pressure (1 mg/kg: 44 ± 3 mm Hg, n = 8; 3 mg/kg 49 ± 2 mm Hg, n = 12), which was followed by a dose-dependent sustained pressor response. The right atrial pressure rose simultaneously and 10–20 s later heart rate decreased. Plasma immunoreactive ANP levels increased significantly (1 mg/kg: 56 ± 10 pmol/1; n = 8; 3 mg/kg: 130 ± 54; n = 12) and also immunoreactive N-terminal ANP levels rose significantly 2 min after the injection of the higher cocaine dose (230 ± 27 pmol/1, n = 12). Significant correlations between plasma immunoreactive ANP levels and all haemodynamic variables were found, especially between mean arterial pressure and plasma immunoreactive ANP levels ( r = 0.86, P < 0.001). In neonatal rat ventricular myocyte cultures, the highest concentration of cocaine (10 −4 M) reduced ANP release into the incubation medium (−41 ± 14%, n = 5) but the reduction was not statistically significant. Our results show that cocaine dose dependently increases ANP and N-terminal ANP secretion into the circulation in conscious rats and that this increase is mediated by haemodynamic changes. Thus, plasma ANP and N-terminal ANP levels could be used as markers for acute cocaine-induced cardiac toxicity.

Phorbol Ester Regulation of Opioid Peptide Gene Expression in Myocardial Cells: ROLE OF NUCLEAR PROTEIN KINASE C

Opioid peptide gene expression was characterized in adult rat ventricular cardiac myocytes that had been cultured in the absence or the presence of phorbol 12-myristate 13-acetate. The phorbol ester induced a concentration- and time-dependent increase of prodynorphin mRNA, the maximal effect being reached after 4 h of treatment. The increase in mRNA expression was suppressed by incubation of cardiomyocytes with staurosporine, a putative protein kinase C inhibitor, and was not observed when the cells were cultured in the presence of the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate. Incubation of cardiac myocytes with phorbol 12-myristate 13-acetate also elicited a specific and staurosporine-sensitive increase in immunoreactive dynorphin B, a biologically active end product of the precursor, both in the myocardial cells and in the culture medium. In vitro run-off transcription assays indicated that transcription of the prodynorphin gene was increased both in nuclei isolated from phorbol ester-treated myocytes and in nuclei isolated from control cells and then exposed to phorbol 12-myristate 13-acetate. No transcriptional effect was observed when cardiac myocytes or isolated nuclei where exposed to 4 alpha-phorbol 12,13-didecanoate. The phorbol ester-induced increase in prodynorphin gene transcription was prevented by pretreatment of myocytes or isolated nuclei with staurosporine, suggesting that myocardial opioid gene expression may be regulated by nuclear protein kinase C. In this regard, cardiac myocytes expressed protein kinase C-alpha, -delta, -epsilon, and -zeta, as shown by immunoblotting. Only protein kinase C-delta and protein kinase C-epsilon were expressed in nuclei that have been isolated from control myocytes, suggesting that these two isotypes of the enzyme may be part of the signal transduction pathway involved in the effect elicited by the phorbol ester on opioid gene transcription in isolated nuclei. The incubation of myocardial nuclei isolated from control cells in the presence of a protein kinase C activator induced the phosphorylation of the myristoylated alanine-rich protein kinase C substrate peptide, a specific fluorescent substrate of the enzyme. The possibility that prodynorphin gene expression may control the heart function through autocrine or paracrine mechanisms is discussed.

Endothelial inhibition of myofilament calcium response in intact cardiac myocytes.

Recent studies suggest that factors released by endothelial cells can modify contraction of isolated cardiac preparations. We compared the effects of 1) coronary effluent collected from Langendorff-perfused rat hearts and 2) cultured vascular endothelial cell superfusate on isolated fura 2-loaded rat ventricular cardiac myocytes. Coronary and cultured cell effluent produced similar effects. Isotonic contraction amplitude was reduced by 31.6 +/- 2.6 and 70.2 +/- 9.1%, respectively; myocyte diastolic length increased by 0.8 +/- 0.2 and 1.5 +/- 0.4 microns, and time to 50% relaxation fell by 6.2 +/- 1.8 and 10.1 +/- 2.0% (all P < 0.05; n = 29 and 15 myocytes, respectively). A small fall in the amplitude of the intracellular Ca2+ transient was observed (8.5 +/- 1.5 and 10.9 +/- 3.5%, respectively; both P < 0.01), insufficient to account for the reduction in twitch amplitude. In intact myocytes tetanized in the presence of thapsigargin, the steady-state myofilament response to Ca2+ was reduced by coronary and cultured cell effluent. These results suggest that both coronary endothelial cells in situ and cultured endothelial cells tonically release a factor(s) that reduces myofilament Ca2+ response.

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart.

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

Dynorphin gene expression and release in the myocardial cell.

The expression of the prodynorphin gene was investigated in adult cultured rat ventricular cardiac myocytes by using a sensitive solution hybridization RNase protection assay for the quantitative analysis of prodynorphin mRNA. Myocyte culture in high KCl resulted, after 4 h, in a marked increase in cellular prodynorphin mRNA, while a KCl treatment for 6, 12, or 24 h progressively down-regulated the levels of prodynorphin mRNA below the control value. Immunoreactive dynorphin B, a biologically active end product of the precursor, was found to be present in the culture medium in significantly higher amounts than in the cardiac myocytes. The levels of this biologically active K opioid receptor agonist significantly increased after 4 h of KCl treatment and were markedly reduced following a 24-h exposure of the cardiac myocytes to KCl. These KCl-induced effects were all abolished by cell incubation in the presence of the calcium channel blocker verapamil. In single cardiac myocytes, acute stimulation of K opioid receptors with dynorphin B or with the selective agonist U-50,488H increased the level of cytosolic calcium. This effect was abolished by the specific K opioid receptor antagonist (Mr-1452) and was not affected by the removal of calcium from the bathing medium. These results suggest that an opioid gene may influence the myocardial function in an autocrine or paracrine fashion.

Prodynorphin mRNA expression in adult cultured rat ventricular cardiac myocytes

Prodynorphin mRNA was synthesized both in rat atrial and ventricular tissue, as well as in adult cultured rat ventricular cardiac myocytes. In the cultured cells, the content of prodynorphin mRNA did not differ from that detected in the original ventricle, indicating that the myocardial cell is an important source for prodynorphin mRNA in the rat ventricular tissue. This study demonstrated the presence of immunoreactive dynorphin B-like material in the cultured cardiomyocytes. Gel permeation chromatography analysis of this material revealed the presence of forms with an apparently higher molecular weight than authentic dynorphin B.

Cyclic AMP metabolism in intact rat ventricular cardiac myocytes: interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine.

Experiments were carried out to elucidate the characteristics of regulation of cyclic AMP levels in intact myocardial cells. For this purpose, the influence of isoproterenol, a nonselective cyclic nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and carbachol on cyclic AMP levels was investigated in isolated rat cardiac myocytes. The extent of cyclic AMP accumulation induced by isoproterenol was much less than that produced by IBMX: submaximal concentrations of isoproterenol and IBMX elevated the cyclic AMP level 2.4- and 4.8-fold of the control level, respectively. Both agents in combination increased the cyclic AMP level markedly 48-fold. Carbachol inhibited the cyclic AMP accumulation induced by isoproterenol, IBMX and their combination by 30%, 60% and 80% of the respective response. The extent of inhibition produced by carbachol of the cyclic AMP accumulation induced by IBMX + isoproterenol was smaller than that caused by propranolol, and carbachol produced only a marginal additional inhibitory action to that of propranolol, implying that carbachol does not affect the process of cyclic AMP degradation. The present findings indicate that in intact cardiac myocytes the rate of cyclic AMP degradation catalyzed by PDE may be a crucial process of cyclic AMP turnover. This view is supported by the observations that the inhibitory action of carbachol on the effect of isoproterenol was less than that on the effect of IBMX, and that the inhibitory action of carbachol was markedly enhanced by the simultaneous presence of IBMX.

HRas-dependent pathways can activate morphological and genetic markers of cardiac muscle cell hypertrophy.

We have investigated the role of the proto-oncogene HRas in cardiac cell growth and hypertrophy. By direct needle microinjection of activated Ras protein into primary neonatal rat ventricular cardiac myocytes, we find that, unlike many other cell types, Ras does not induce DNA synthesis in these cells. However, injection of activated Ras does induce expression of both the c-Fos and atrial natriuretic factor (ANF) genes. Expression of both these genes is associated with the hypertrophic response in ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling pathway. Ras injection also causes morphological changes in the cells so that they increase in profile and show changes in the organization of the contractile apparatus. Further support for a role for Ras in the hypertrophic response was obtained from studies showing that activated Ras stimulates ANF promoter activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the hypertrophic stimulation of the ANF promoter by phenylephrine, indicating a role for Ras in the hypertrophic effect of an alpha-adrenergic agonist.

Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.

1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac myocytes. Intracellular pH was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura-2. 2. Application of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 +/- 34% (n = 8) (p less than 0.02). The rate of change of cell length during contraction (i.e. +dL/dt) increased from 67.2 +/- 8.7 microns/s to 127.7 +/- 14.1 microns/s (n = 7). The rate of change of cell length during relaxation (-dL/dt) increased from 55.8 +/- 7.4 microns/s to 118.9 +/- 12.1 microns/s (n = 7). Time to peak shortening was unchanged. 3. Application of 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7.5 +/- 7.5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C. 4. In rat myocytes we have measured the changes of pHi and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p less than 0.05) enhanced by 15.1 +/- 6.9% (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p less than 0.05) by an average of 59.1 +/- 10.6% (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4-phorbol 12,13-didecanoate (which does not activate protein kinase C). 5. Since pHi recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM-amiloride and inhibited by removing extracellular Na+ then it is suggested that, like pHi regulation in sheep heart Purkinje fibres, pHi recovery in rat cardiac ventricular myocytes is mainly through sarcolemmal Na(+)-H+ exchange. We suggest that in the presence of TPA the Na(+)-H+ exchange is stimulated. 6. The relationship between pHi and cell shortening is non-linear as has been observed by others in whole tissue preparations. The presence of TPA shifts the relationship upwards such that at any one pHi, cell shortening is greater.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison between alpha-adrenergic- and K-opioidergic-mediated inositol(1,4,5)P 3/inositol(1,3,4,5)P 4 formation in adult cultured rat ventricular cardiomyocytes

In adult cultured rat ventricular cardiac myocytes, both the α-adrenergic agonist phenylephrine and the selective K opioid receptor ligand U-50,488H affected phosphoinositide turnover. Phenylephrine, over a time course of 10 min, caused a transient increase in Ins(1,4,5)P 3 which peaked at 1 min and had returned to control at 2 min. In addition, phenylephrine produced a progressive and sustained increase in the formation of Ins(1,3,4, 5)P 4 which achieved a plateau after 5 min of exposure to the agonist. U-50,488H induced an increase in Ins(1,4,5)P 3 which peaked at 1 min at a level significantly higher than that due to phenylephrine and was still elevated after 10 min exposure to the K opioid receptor agonist. In addition, U-50,488H caused a sustained increase in Ins(1,3,4,5)P 4 which was comparable to that due to phenylephrine. The stimulatory effects produced by phenylephrine and U-50,488H were receptor-mediated events, since they were fully antagonized by their respective antagonists, phentolamine or Mr-1452.

Cyclic AMP-dependent regulation of the number of [3H]batrachotoxinin benzoate binding sites on rat cardiac myocytes

We sought to assess the effect of an increase in cAMP on sodium channels on adult rat cardiac ventricular myocytes. Sodium channels were studied with the use of the radiolabeled sodium channel-specific toxin [3H] batrachotoxinin benzoate ([3H]BTXB). Forskolin, isoproterenol, prostaglandin E1, cholera toxin, and pertussis toxin each increased cAMP levels and decreased the number of [3H]BTXB binding sites without changing the affinity of [3H]BTXB for the sodium channel. The cAMP analog 8-bromo-cyclic AMP (8-Br-cAMP) reduced the number of [3H]BTXB binding sites from 19 fmol/10(5) cells to 11 fmol/10(5) cells. [3H]BTXB binding site down-regulation was reversible, cAMP dose-dependent, and time-dependent. To test the hypothesis that the cAMP effect was mediated by cAMP-dependent phosphorylation, we determined the effect of 8-Br-cAMP on [3H]BTXB binding after preincubation of myocytes with N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide dihydrochloride (H8), a protein kinase A inhibitor. H8 inhibited 70% of the decrease in the number of [3H]BTXB binding sites induced by 8-Br-cAMP. Thus increases in intracellular cAMP in cardiac myocytes reversibly induced a decrease in the number of [3H]BTXB binding sites via cAMP-dependent protein phosphorylation, possibly of the sodium channel.

Local activation of contraction in isolated rat ventricular myocytes

The aim of this paper was to examine whether contraction (which is thought to result from Ca-induced release of Ca2+ from the sarcoplasmic reticulum) can propagate along a cardiac cell. The experiments were performed on isolated rat ventricular cardiac myocytes. Two techniques were used to initiate contraction in a localized region of the cell. 1) The cells were loaded with the "caged" Ca-containing compound nitr-5. A region of the cell was illuminated with ultraviolet light to increase intracellular Ca2+ concentration ([Ca2+]i) in that area. 2) The cells were superfused with a low (0.1 mM) Ca solution to abolish contraction. Ca was applied to a region of the cell by iontophoresis, and the cell was then electrically stimulated. In both cases a local contraction was produced that did not spread to the rest of the cell. Like the normal twitch, the local contraction was abolished by ryanodine, showing that it is produced by Ca release from the sarcoplasmic reticulum. In the final series of experiments, the cell was stimulated to contract in a control (1 mM Ca2+) solution. Cd2+ was then iontophoresed to a region of the cell. Only the region of the cell exposed to Cd stopped contracting. In conclusion, the above results show that the systolic rise of [Ca2+]i cannot propagate through a cell. This lack of propagation is suggested to be important in stopping a rise of [Ca2+]i in one cell spreading to the rest of the heart.

Identification of P2Y purinoceptors associated with voltage‐activated cation channels in cardiac ventricular myocytes of the rat

Extracellular ATP has vasodilatory and inotropic effects in the heart. We have demonstrated that extracellular ATP, in a concentration-dependent manner (10 nM-0.1 mM), increased [Ca2+]i in suspensions of isolated fura-2-loaded rat cardiac ventricular myocytes (maximum 96 +/- 10% increase over basal levels, SEM, n = 12, P less than 0.01). The increase in [Ca2+]i was often biphasic, with an initial fast phase (less than 1 s) of low amplitude, followed by a slower phase of higher amplitude. A second application of ATP had little effect, and ATP abolished the effect of subsequent electrical stimulations, even through the cells were still able to respond with an increase in [Ca2+]i to KCl-induced depolarization or stimulation by caffeine. Pretreatment of cells with nifedipine, verapamil, caffeine, ryanodine, or 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride attenuated the effect of extracellular ATP on [Ca2+]i, and binding of extracellular free calcium by excess EGTA completely abolished the effects of extracellular ATP and electrical stimulation. Extracellular ATP increased bisoxonol fluorescence in ventricular myocytes, indicating depolarization of the sarcolemma. Pretreatment of the myocytes with tetrodotoxin (50 microM), or replacement of NaCl in the incubation buffer with the impermeant cation N-methyl-D-glucamine, suppressed the extracellular ATP effect on [Ca2+]i. ADP and AMP had smaller effects on [Ca2+]i than ATP; adenosine had no effect. ATP analogues showed the following rank order of potency in increasing [Ca2+]i or bisoxonol fluorescence: ATP greater than or equal to 2-methylthioATP much greater than adenosine 5'-O-[3-thio]triphosphate greater than adenosine 5'-[alpha, beta-methylene]triphosphate approximately adenosine 5'-[beta, gamma-methylene]triphosphate approximately adenosine 5'-[beta, gamma-imino]triphosphate greater than adenosine. These data are consistent with the presence of purinoceptors (P2Y subtype) on the sarcolemma of cardiac ventricular myocytes of the rat, which upon activation lead to depolarization and activation of cation channels of the sarcolemma and flux of extracellular Ca2+ into the cells. This may result in further flux of Ca2+ into the cytosol from intracellular stores. The effects of extracellular ATP on [Ca2+]i in rat cardiac ventricular myocytes may, in part, explain the direct inotropic effects of extracellular ATP on the mammalian heart.

Decrease with senescence in the norepinephrine-induced phosphorylation of myofilament proteins in isolated rat cardiac myocytes

(1) The effects of norepinephrine on protein phosphorylation in isolated rat cardiac ventricular myocytes were determined by autoradiography of 32P-labelled proteins separated by electrophoresis; (2) In cells from young adult rats (6 months old) there was a marked increase due to norepinephrine (10 −8 to 10 −4 m) in the incorporation of 32P into proteins identified on the grounds of molecular weight as troponin I and C-protein: in cells from senescent rats (24 months old) this increase was much attenuated. (3) Age-associated decrements in protein phosphorylation were much diminished when maximally effective concentrations of the adenylate cyclase-activator forskolin and the cyclic AMP analog 8(4-chlorophenylthio) cyclic AMP were used instead of norepinephrine. Moreover, age-associated differences were abolished if the phosphodiesterase inhibitor isobutylmethylxanthine was present in addition to norepinephrine, or alone. (4) Study of the rates of dephosphorylation of troponin I, as initiated with the β-adrenergic antagonist propranolol, showed no change in half-time as a function of age: this indicates no change in protein phosphatase activity. (5) These results suggest that there is less active net formation of cyclic-AMP in senescent heart cells in response to the neurotransmitter norepinephrine, giving a lesser activation of c-AMP-dependent protein kinase and less phosphorylation of these target proteins.

ATP receptor-induced Ca2+ transients in cardiac myocytes: sources of mobilized Ca2+

Addition of micromolar concentrations of extracellular ATP to adult rat cardiac ventricular myocytes increases cytosolic Ca2+ concentration ([Ca2+]). Experiments were performed on fura-2-loaded myocytes to determine whether the [Ca2+] rise was due to Ca2+ influx, release of Ca2+ from the sarcoplasmic reticulum (SR), or a combination of both. BAY K 8644 and nifedipine affected ATP-induced [Ca2+] transients, indicating involvement of voltage-sensitive Ca2+ channels. Addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+ channel blockers significantly reduced cytosolic [Ca2+] changes due to addition of ATP or KCl without depleting Ca2+ stores (shown by ionomycin treatment in a Ca2+-free medium), demonstrating that these responses require Ca2+ influx. Depletion of intracellular Ca2+ stores by caffeine or ryanodine also diminished cytosolic [Ca2+] responses, indicating that a portion of the increased cytosolic [Ca2+] is due to Ca2+ release from SR. Norepinephrine potentiates the ATP-Ca2+ response, and this effect was not inhibited by depletion of intracellular stores. Although the data show that there are two Ca2+ sources in the cytosolic Ca2+ response to ATP, the pattern is also consistent with the hypothesis of Ca2+-induced Ca2+ release from cardiac SR.