Cellular therapies for myocardial infarct repair.
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Animals↗
Biomedical subjects
M A Laflamme
Publications and source records attributed to M A Laflamme.
G(s) and adenylyl cyclase in transverse tubules of heart: implications for cAMP-dependent signaling.
The transverse tubules are highly specialized invaginations of the cardiac sarcolemmal membrane involved in excitation-contraction (EC) coupling. Several proteins directly involved in EC coupling have been shown to reside either in the transverse tubular membrane or in closely associated structures. With the use of immunofluorescence microscopy, we have found that G(S) and adenylyl cyclase, key elements in the beta-adrenergic signal transduction cascade, are essentially homogeneously distributed throughout the transverse tubular network of isolated rat ventricular myocytes. G(S), in particular, was much more abundant within the transverse tubular membrane than in the peripheral sarcolemma. Furthermore, both proteins are also present in the intercalated disk region. The location of these elements of the cAMP-signaling cascade within a few micrometers of every inotropic target suggests that control and action of this second messenger are quite local. Furthermore, a similar distribution is likely for negatively inotropic receptor systems that oppose G(S)-linked receptors at the level of adenylyl cyclase. Thus, in addition to their role in EC coupling, transverse tubules appear to be the primary site for signaling by inotropic agents.
Adenylyl Cyclases↗
Do beta 2-adrenergic receptors modulate Ca2+ in adult rat ventricular myocytes?
We examined the role of beta 2-adrenergic receptors (ARs) in modulating calcium homeostasis in rat ventricular myocytes. Zinterol (10 microM), an agonist with a 25-fold greater affinity for beta 2-ARs over beta 1-ARs, modestly enhanced L-type calcium current (ICa) magnitude by approximately 30% and modestly accelerated the rate of Ca2+ concentration ([Ca2+]) decline (approximately 35%) but had little effect on the magnitude of the [Ca2+] transient (a nonsignificant 6% increase). However, 1 microM of the highly selective beta 1-AR antagonist CGP-20712A completely blocked the ICa increase induced by 10 microM zinterol. Pretreatment of cells with pertussis toxin (PTX) did not alter ICa enhancement by 10 microM zinterol, although it did abolish the ability of acetylcholine to block the forskolin-induced enhancement of ICa. Zinterol (10 microM) approximately doubled adenosine 3',5'-cyclic monophosphate (cAMP) accumulation, although one-half of this increase was blocked by CGP-20712A. In contrast, 1 microM of the nonselective beta-agonist isoproterenol increased cAMP production 15-fold. Thus we found no evidence that activation of beta 2-ARs modulates calcium homeostasis in rat ventricular myocytes, even after treatment with PTX.
Adrenergic beta-Agonists↗
Ca2+-induced current oscillations in rabbit ventricular myocytes.
Transient currents are activated by spontaneous Ca2+ oscillations in rabbit ventricular myocytes. We investigated the ionic basis for these transient currents under conditions in which K+ currents would be expected to be blocked. Holding cells under voltage clamp at positive potentials leads to a rise in intracellular Ca2+ via reversal of the Na+-Ca2+ exchanger and subsequently to the initiation of spontaneous Ca2+ transients, presumably from a Ca2+-overloaded sarcoplasmic reticulum. The current transients associated with these Ca2+ transients reversed at about +10 to +15 mV under conditions of approximately symmetrical Cl-. In the absence of Cl-, this current was inward at all potentials examined over the range from -88 to +72 mV, consistent with a Na+-Ca2+ exchanger current. In the absence of Na+, the repetitive spontaneous Ca2+ transients could be initiated by a brief train of depolarizations to activate the inward Ca2+ current. Under such conditions, the current was found to reverse at -3 mV when the equilibrium potential of Cl- (ECl) was -2 mV, and the reversal potential shifted to -32 mV when internal Cl- was lowered, to make ECl -33 mV. Thus, in the absence of Na+, it appears that the current is exclusively a Ca2+-activated Cl- current. There is no evidence to indicate the presence of a Ca2+-activated cationic conductance. Further, our results demonstrate that the Ca2+-activated Cl- conductance can carry inward current at potentials more negative to ECl in rabbit ventricular myocytes and is therefore likely to contribute to the arrhythmogenic delayed afterdepolarizations that occur in Ca2+-overloaded cells.
Animals↗
Cyclic ADP-ribose does not regulate sarcoplasmic reticulum Ca2+ release in intact cardiac myocytes.
Cyclic ADP-ribose (cADPR), an intracellular second messenger known to mobilize Ca2+ in sea urchin eggs, has been implicated in modulating Ca2+ release in a variety of mammalian tissues. On the basis of studies of isolated cardiac sarcoplasmic reticulum (SR) vesicles and single SR Ca2+ release channels, cADPR has also been proposed to be a modulator of SR Ca2+ release in heart. In the present study, we directly examined the ability of cADPR to trigger SR Ca2+ release and to modulate Ca(2+)-induced Ca2+ release (CICR) in intact rat ventricular myocytes. Voltage-clamped myocytes were dialyzed with up to 100 mumol/L caged cADPR and 0.6 mumol/L calmodulin along with the Ca(2+)-sensitive dye fluo 3. A step increase in the cADPR concentration induced by flash photolysis of caged cADPR neither directly triggered SR Ca2+ release nor modulated CICR in intact myocytes. In contrast, under similar conditions, extracellular application of caffeine (1 to 2.5 mmol/L) onto myocytes produced both effects. Under equivalent conditions, flash photolysis of caged cADPR-loaded sea urchin eggs resulted in large Ca2+ transients. Further, the sustained presence of high cytosolic concentrations of either cADPR or its antagonist, 8-amino-cADPR, was ineffective in altering normal CICR in myocytes. These findings indicate that cADPR does not regulate SR Ca2+ release in intact cardiac myocytes.
Adenosine Diphosphate Ribose↗