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.

In atrial myocytes, an initial exposure to isoproterenol (ISO) acts via cAMP to mediate a subsequent acetylcholine (ACh)-induced activation of ATP-sensitive K+ current (IK,ATP). In addition, β-adrenergic receptor (β-AR) stimulation activates nitric oxide (NO) release. The present study determined whether the conditioning effect of β-AR stimulation acts via β1- and/or β2-ARs and whether it is mediated via NO signaling. 0.1 μM ISO plus ICI 118,551 (ISO-β1-AR stimulation) or ISO plus atenolol (ISO-β2-AR stimulation) both increased L-type Ca2+ current (ICa,L) markedly, but only ISO-β2-AR stimulation mediated ACh-induced activation of IK,ATP. 1 μM zinterol (β2-AR agonist) also increased ICa,L and mediated ACh-activated IK,ATP. Inhibition of NO synthase (10 μM L-NIO), guanylate cyclase (10 μM ODQ), or cAMP-PKA (50 μM Rp-cAMPs) attenuated zinterol-induced stimulation of ICa,L and abolished ACh-activated IK,ATP. Spermine-NO (100 μM; an NO donor) mimicked β2-AR stimulation, and its effects were abolished by Rp-cAMPs. Intracellular dialysis of 20 μM protein kinase inhibitory peptide (PKI) abolished zinterol-induced stimulation of ICa,L. Measurements of intracellular NO ([NO]i) using the fluorescent indicator DAF-2 showed that ISO-β2-AR stimulation or zinterol increased [NO]i. L-NIO (10 μM) blocked ISO- and zinterol-induced increases in [NO]i. ISO-β1-AR stimulation failed to increase [NO]i. Inhibition of Gi-protein by pertussis toxin significantly inhibited zinterol-mediated increases in [NO]i. Wortmannin (0.2 μM) or LY294002 (10 μM), inhibitors of phosphatidylinositol 3′-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated IK,ATP and stimulate [NO]i. We conclude that both β1- and β2-ARs stimulate cAMP. β2-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via Gi-protein and PI-3K coupled to NO-cGMP signaling. Only β2-ARs acting exclusively via NO signaling mediate ACh-induced activation of IK,ATP. NO signaling also contributes to β2-AR stimulation of ICa,L. The differential effects of β1- and β2-ARs can be explained by the coupling of these two β-ARs to different effector signaling pathways.

Whole‐cell recording methods and fluorescence microscopy were used to study the effects of acute exposure to thyroid hormone (T3) on cat atrial myocytes. Acute exposure (≈5 min) to 10 nm T3 significantly increased tetrodotoxin (TTX)‐sensitive inward Na+ current (INa) at voltages between −40 and +20 mV. At maximal INa activation (−40 mV) T3 increased peak INa by 32 %. T3 had no effect on the time course of INa decay, voltage dependence of activation, inactivation, or recovery from inactivation. Comparable exposures to reverse T3 (rT3) or T4 had no effect on INa. L‐type Ca2+ current was unaffected by acute exposure to T3. T3‐induced increases in INa were unaffected by 50 μm nickel, a blocker of T‐type Ca2+ current. T3 significantly increased cell shortening (+62 %) and could elicit spontaneous action potentials arising from Ca2+‐mediated after‐depolarizations. T3 (but not rT3) significantly increased baseline intracellular Ca2+, release of Ca2+ from sarcoplasmic reticulum (SR) and caffeine (10 mm)‐induced release of SR Ca2+. We conclude that acute T3 exposure increases Na+ influx via INa and thereby stimulates reverse‐mode Na+‐Ca2+ exchange to increase intracellular Ca2+ content and release. As a result, T3 increases contraction strength, and can initiate Ca2+‐mediated arrhythmic activity. Acute non‐genomic effects of T3 can contribute to the positive inotropy and sinus (atrial) tachycardia traditionally attributed to chronic, genomic effects of elevated thyroid hormone on atrial muscle.

A perforated patch recording method was used to determine the effects of genistein (Gen), a protein tyrosine kinase (PTK) inhibitor, on basal L-type Ca2+ current ( I Ca,L) in feline atrial myocytes. Gen (50 μM) elicited biphasic changes in I Ca,L: an initial inhibition (−55 ± 4%; phase 1) followed by a secondary stimulation (34 ± 9%; phase 2) of I Ca,L. Withdrawal of Gen elicited a further potentiation of I Ca,L (152 ± 19%; phase 3) above control ( n = 46). In general, phase 1 inhibition and phase 3 potentiation varied directly with Gen concentration, and phase 2stimulation exhibited biphasic concentration-dependent changes compared with control. When cells were dialyzed using a ruptured patch recording method, Gen elicited only inhibition of I Ca,L; phases 2 and 3 were abolished. Vanadate (1 mM), an inhibitor of protein tyrosine phosphatase, abolished both Gen-induced inhibition and stimulation of I Ca,L. Daidzein (50 μM), a weakly active analog of Gen, exerted no significant effects on I Ca,L, and withdrawal of daidzein failed to potentiate I Ca,L. In a few cells, Gen elicited a prominent vanadate-sensitive stimulation of I Ca,L in the absence of any significant inhibition of I Ca,L. Gen-induced changes in I Ca,L were unaffected by either 100 μM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM) or 1 μM ryanodine, agents that alter intracellular Ca2+; 4 μM H-89 or 50 μM Rp diastereomer of adenosine 3′,5′-monophosphothioate (RP-cAMPS), inhibitors of protein kinase A (PKA); 0.1 μM calphostin C or 2 μM chelerythrine, inhibitors of protein kinase C (PKC); or 100 μM N G-monomethyl-l-arginine (l-NMMA), an inhibitor of nitric oxide (NO) synthase. We conclude that in feline atrial myocytes, Gen acts via membrane-bound PTKs to inhibit I Ca,L and via cytosolic PTKs to stimulate I Ca,L. Gen-induced changes in I Ca,L are not related to changes in intracellular Ca2+ or to secondary interactions with either PKA, PKC, or NO signaling pathways. These results indicate that in atrial myocytes I Ca,L is regulated by two independent and competing PTK signaling mechanisms.

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