In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.

A perforated-patch whole-cell recording method was used to determine whether nitric oxide signaling participates in acetylcholine (ACh)-induced regulation of basal L-type Ca 2� current (I Ca,L ) in cat atrial myocytes. Exposure to 1 �M ACh for 2 min inhibited basal I Ca,L (�21 � 3%), and withdrawal of ACh elicited rebound stimulation of I Ca,L above control (80 � 13%) (n � 23). Stimulation of I Ca,L elicited by withdrawal of ACh (but not ACh-induced inhibition of I Ca,L ) was blocked by either 50 �M hemoglobin; 30 �M ODQ or 10 �M methylene blue, inhibitors of soluble guanylate cyclase; 10 �M W-7, a calmodulin inhibitor; or 10 �M L-NIO, an inhibitor of constitutive NO synthase (NOS). In cells incubated in 5 mM L-arginine, ACh-induced rebound stimulation of I Ca,L was enhanced compared with control responses. Histochemical assay (NADPH diaphorase) indicated that atrial myocytes express constitutive NOS. NO-donor, spermine/NO (SP/NO), �1 �M stimulated basal I Ca,L . SP/NO-induced stimulation of I Ca,L was inhibited by 50 �M hemoglobin, 30 �M ODQ, or 5 �M H-89, an inhibitor of PKA, and was unchanged by 50 �M MnTBAP, a peroxynitrite scavenger. When I Ca,L was prestimulated by 10 �M milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase I Ca,L . In cells incubated in pertussis toxin (3.4 �g/ml for 6 h; 36�C), ACh failed to affect I Ca,L , but 100 �M SP/NO or 10 �M milrinone still increased basal I Ca,L . These results indicate that in cat atrial myocytes NO signaling mediates stimulation of I Ca,L elicited by withdrawal of ACh but not ACh-induced inhibition of basal I Ca,L . NO activates cGMP-induced inhibition of phosphodiesterase (type III) activity. Upon withdrawal of ACh, this mechanism allows cAMP to recover to levels above control, thereby stimulating I Ca,L . Pertussis toxin-sensitive G-proteins couple M 2 muscarinic receptors to NO signaling. NO-mediated stimulation of I Ca,L elicited by withdrawal of ACh may be an important mechanism that rapidly restores cardiac pacemaker and contractile functions after cholinergic suppression of atrial activity.