A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium

Powell, T.; Twist, V. W.

doi:10.1016/0006-291x(76)90997-9

Cited by 290 publications

(119 citation statements)

References 11 publications

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“…Animals were kept at the animal house on a normal laboratory diet and tap water ad libitum. Cells were obtained by enzymatic dispersion of hamster ventricle after the method of Powell and Twist 15 and Tanigushi et al 16 The heart was removed and perfused immediately with normal Krebs solution containing the following (in mmol/L): 136.5 NaCl, 5.4 KCl, 1.8 CaCl 2 , 0.53 MgCl 2 , 0.3 NaH 2 PO 4 , 11.9 NaHCO3, 5.5 glucose, and 5 HEPES, with pH adjusted to 7.3. After 20 minutes, a calcium-free solution containing 0.4% collagenase (Worthington) was recirculated through the heart for 1 hour.…”

Section: Methodsmentioning

confidence: 99%

Intracellular and Extracellular Angiotensin II Enhance the L-Type Calcium Current in the Failing Heart

Mello

Monterrubio

2004

Hypertension

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Abstract-The influence of intracellular and extracellular angiotensin II (Ang II) on the L-type calcium current of cardiomyocytes isolated from cardiomyopathic hamsters was investigated. The results indicated that Ang II (10 Ϫ8 mmol/L), added to the bath, increased the peak inward calcium current (I Ca ) density by 37Ϯ3.4% (PϽ0.05), an effect that depends on the activation of protein kinase C. Intracellular administration of the same dose of Ang II (10 Ϫ8 mmol/L) also elicited an increase of peak I Ca density but enhanced the rate of I Ca inactivation, an effect not seen with extracellular Ang II. Moreover, in control animals, no change in the rate of I Ca inactivation was seen with intracellular Ang II. Thapsigargin (1 mol/L), a potent inhibitor of sarcoplasmic reticulum (SR) ATPase, which depletes the SR, decreased the rate of I Ca inactivation elicited by intracellular Ang II, although the cytoplasmic calcium concentration was highly buffered with 10 mmol/L EGTA. These findings might indicate that intracellular Ang II releases calcium from the SR and inactivates I Ca . The effect of intracellular Ang II on peak I Ca was not altered by extracellular losartan (10 Ϫ7 mmol/L), supporting the notion that the peptide acted intracellularly. Other studies showed that intracellular Ang I administration (10 Ϫ8 mmol/L) enhanced the peak I Ca density and the rate of I Ca inactivation, an effect that was reduced by intracellular enalaprilat (10 Ϫ8 mmol/L). Moreover, intracellular enalaprilat by itself reduced the peak I Ca density. These observations might indicate that endogenous Ang II is contributing to I Ca modulation in the failing heart. Key Words: angiotensin Ⅲ heart failure Ⅲ calcium current T he concept of a cardiac renin-angiotensin system 1 gained support with the demonstration that: (1) angiotensin I (Ang I) is converted to Ang II in the isolated and perfused heart; 2 (2) the angiotensin-converting enzyme (ACE) has been found around the nucleus of heart cells in culture 3 ; and (3) ACE inhibitors prevent cardiac remodeling, an effect independent of the change in blood pressure. 4 However, in normal heart, renin mRNA levels 5 and renin content are negligible in nephrectomized rats. 6 Moreover, no renin is released from the isolated and perfused rat heart, 7 which suggests that cardiac renin is attributable to its uptake from plasma. However, under some conditions, such as stretch, renin gene expression is enhanced, 8 and overexpression of angiotensinogen gene in normal mice leads to hypertrophy of the right and left ventricles and to an increase of Ang II levels in both ventricles without any change in arterial blood pressure. 9,10 Furthermore, a second renin gene transcript that may code for intracellular renin because it lacks the coding zone of the secretory signal peptide is upregulated in the left ventricle after myocardial infarction. 11 Moreover, cardiac angiotensinogen is upregulated after myocardial infarction. 12 These observations support the notion that renin and Ang II can be formed inside the hear...

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Section: Methodsmentioning

confidence: 99%

Intracellular and Extracellular Angiotensin II Enhance the L-Type Calcium Current in the Failing Heart

Mello

Monterrubio

2004

Hypertension

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“…Hearts were retrogradely perfused and equilibrated with a modified Krebs Ringer solution (KRB), containing (mM): NaCl 110, KCI 2.6, NaHCO3 25, MgSO4 1.2, KH2PO4 1.2 and glucose 11 (pH 7.4), which had just previously been gassed with 95% 02/5% CO2 at 37°C. After the initial period of washout, collagenase was added to the perfusate at a concentration of 0.04% (w/v), which was recirculated through the heart in the presence of low levels of CaCl2 (25 fiM) for 20-25 min (Powell & Twist, 1976 …”

Section: Methodsmentioning

confidence: 99%

Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes

Kelso

McDermott

Silke

1993

British J Pharmacology

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1 The contractile effects of the novel cardiotonic agent HN-10200 (2-[3-methoxy-5-methylsulphinyl-2-thienyl]-lH-imidazo-[4,5-c]-pyridine hydrochloride), were examined and comparisons made with the responses obtained to a structurally similar compound, sulmazole, and to a number of other compounds which are known to inhibit phosphodiesterase (PDE) isoenzymes with differing selectivities; namely, enoximone (PDE III inhibitor), Ro 20-1724 (PDE IV inhibitor) and 3-isobutyl-1-methylxanthine (nonselective PDE inhibitor). 2 Contractile function, as measured by mechanical shortening, and biochemical systems involving cyclic AMP were investigated in ventricular cardiomyocytes isolated from adult Sprague-Dawley rats (200-250 g). 3 HN-10200 exerted a concentration-dependent (10-8 M-10-4 M) positive contractile effect, which was independent of a-or 0-adrenoceptor, or histamine receptor stimulation. 4 The efficacies of the contractile responses to the PDE inhibitors were of the order: HN-10200> IBMX> sulmazole> enoximone and maximum stimulations, which were obtained at concentrations of 10-4M, were 54 ± 4%, 41 ± 7%, 38 ± 7% and 26 ± 5% (mean ± s.e.) greater than basal levels, respectively (n = 6); the basal value of contractile amplitude (dL), in the absence of PDE inhibitors was 7.39 ± 0.18% (mean ± s.e.). Ro 20-1724 did not have any effect on contractile activity. 5 Due to low basal levels of cyclic nucleotides in isolated cells, accumulation of cyclic AMP due to the presence of the PDE inhibitors was detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 fM). 6 The PDE inhibitors increased levels of cyclic AMP only at concentrations> 10-' M. HN-10200 and sulmazole had similar concentration-dependent profiles for the accumulation of cyclic AMP; their potencies were lower than that of IBMX (concentrations of forskolin required to increase cyclic AMP by 4 pmol mg-' protein, in the presence of maximum concentrations of the PDE inhibitors, were 13 ± 3 11M, 14 ± 3 JAM and 3 ± 0.6 JAM [mean ± s.e.], respectively). 7 These results indicate that a similar mechanism, probably through a weak inhibition of the cyclic AMP-specific PDE isoenzymes, is responsible for the increase in levels of cyclic AMP by HN-10200 and sulmazole. However, cyclic AMP is only partially responsible for the positive contractile effect of HN-10200 and, similarly, sulmazole and IBMX. The lack of apparent increase in levels of cyclic AMP by enoximone, highlights its degree of selectivity for the PDE III isoenzyme, such that the PDE IV isoform is still present in sufficient quantity to degrade cyclic AMP within the cell. On the other hand, the potent action of Ro 20-1724 on accumulation of cyclic AMP, in addition to the lack of effect on contractile function, is in agreement with the selectivity of this compound for the PDE IV isoenzyme and compartmentalization of cyclic AMP in rat isolated ventricular cardiomyocytes.

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“…These were obtained by an enzymatic dispersion of the heart (essentially based on the method of Powell & Twist, 1976). Briefly, the heart is rapidly removed from a phenobarbitone-anaesthetized guinea-pig (of either sex, weighing 400-700g).…”

Section: Methodsmentioning

confidence: 99%

“…Specifically, new methods of cell isolation (Powell & Twist, 1976) have enabled the investigation of membrane currents in single cardiac cells, without complications in interpretation which were inherent in previous work on multicellular cardiac preparations. New and better methods of voltage clamping such isolated cells also provide more reliable information about the membrane currents which underlie the electrical and mechanical activity of the heart.…”

Section: Introductionmentioning

confidence: 99%

The effects of prenylamine on single ventricular myocytes of guinea‐pig

Shimoni

Posner²,

Spindler³

et al. 1988

British J Pharmacology

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1 The action of prenylamine, an antianginal drug, was studied in single ventricular guinea-pig myocytes. In concentrations of 10-50 pm, prenylamine significantly (P <0.01) shortened action potentials, and significantly (P < 0.001) reduced the inward calcium current by 29% to 76% (n = 7). This effect was also present in the presence of adrenoceptor-blockade (with phentolamine and propranolol), and was thus not due to indirect changes in endogenous catecholamine action. 2 Prenylamine did not affect the steady state level of current at the end of long pulses, and does therefore not act by changing time-dependent outward currents. Since the resting potential in the unclamped mode is unchanged during gross changes in action potential duration, it is also unlikely that there are any changes in the background, time-independent potassium conductance. 3 It is concluded that prenylamine has a direct effect on cardiac calcium channels, not mediated by adrenoceptor activation.

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A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium

Cited by 290 publications

References 11 publications

Intracellular and Extracellular Angiotensin II Enhance the L-Type Calcium Current in the Failing Heart

Intracellular and Extracellular Angiotensin II Enhance the L-Type Calcium Current in the Failing Heart

Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes

The effects of prenylamine on single ventricular myocytes of guinea‐pig

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