Ca²⁺‐stimulated adenylyl cyclase isoform AC1 is preferentially expressed in guinea‐pig sino‐atrial node cells and modulates the I_f pacemaker current

Abstract

Ca2+ -stimulated adenylyl cyclases (AC) are known to play important roles in neurons but have not previously been reported in the heart. Here we present the first evidence for selective expression of Ca 2+ -stimulated AC in the sino-atrial node (SAN) but not in ventricular muscle of the guinea-pig heart. The AC1 isoform of Ca 2+ -stimulated AC was shown to be present in SAN, both as mRNA using RT-PCR and as protein using immuno-blotting with a specific antibody. Confocal immuno-fluorescence studies detected membrane localization of AC1 in SAN cells, but no AC1 in ventricular muscle. Ca 2+ -stimulated AC8 may also be present in SAN. The functional importance of AC activity was investigated by monitoring activation of I f (gated by hyperpolarization and regulated by cAMP, which shifts activation to more depolarized voltages). Basal activity of AC in isolated SAN myocytes was demonstrated by the observations that an inhibitor of AC activity (MDL 12330A, 10 μM) shifted activation in the hyperpolarizing direction, while inhibition of phosphodiesterases (IBMX, 100 μM) shifted I f activation in the depolarizing direction. Buffering cytosolic Ca 2+ with the Ca 2+ chelator BAPTA (by exposure to BAPTA-AM) shifted activation of I f in the hyperpolarizing direction, and under these conditions the AC inhibitor MDL had little or no further effect. The actions of BAPTA were overcome by exposure to forskolin (10 μM), a direct stimulator of all AC isoforms, to restore cAMP levels. These effects are consistent with the functional importance of Ca 2+ -stimulated AC, which is expected to be fundamental to initiation and regulation of the heartbeat.

Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

“…Figure 2A shows the increase in spontaneous AP firing rate in response to these perturbations: 21.9 Ϯ 1.1% by ␤-AR stimulation with ISO (1 M) (n ϭ 9), 25.6 Ϯ 2.2% by PDE inhibition by IBMX (100 M) (n ϭ 9), 24.6 Ϯ 2.7% by phospho-protein phosphatase inhibition by calyculin A (1 M) (n ϭ 10), and 22.6 Ϯ 3.9% by addition of a membrane-permeable cAMP analog, CPTcAMP (n ϭ 9). The increases in spontaneous AP firing rate induced by these pharmacologic interventions are similar to those measured in prior studies (1,7,15,16,27,37,44).…”

“…P Ͻ 0.05 was taken to indicate statistical significance. (15,27,37), protein phosphatase inhibition (1,44), or an increase in cAMP (7) increase the spontaneous AP firing rate in single SANC. Figure 2A shows the increase in spontaneous AP firing rate in response to these perturbations: 21.9 Ϯ 1.1% by ␤-AR stimulation with ISO (1 M) (n ϭ 9), 25.6 Ϯ 2.2% by PDE inhibition by IBMX (100 M) (n ϭ 9), 24.6 Ϯ 2.7% by phospho-protein phosphatase inhibition by calyculin A (1 M) (n ϭ 10), and 22.6 Ϯ 3.9% by addition of a membrane-permeable cAMP analog, CPTcAMP (n ϭ 9).…”

“…1), i.e., surface membrane electrogenic proteins, functioning as a voltage oscillator ("membrane clock"), and sarcoplasmic reticulum (SR) ( (2,19). This high-throughput Ca 2ϩ -calmodulin-activated AC/PKA-CaMKII-Ca 2ϩ release signaling cascade in pacemaker cells is regulated in a feed-forward manner (27,43) and requires physiological "brakes" to maintain the basal spontaneous action potential (AP) firing rate well below its maximum (Fig. 1).…”

“…1). This regulation is accomplished by basal phosphodiesterase (PDE) (15,27,37) and protein phosphatase activity (1,44) (Fig. 1).…”

The A-kinase anchoring protein Yotiao binds and regulates adenylyl cyclase in brain

“…Normal I(Ks) channel activity is stimulated by sympathetic activity in cardiac myocytes (12). In the heart, AC 2 and 3 are expressed in cardiac fibroblasts, whereas AC1 has been reported only in the sino-atrial node (21,22). AC3 is present mainly in the developing heart with decreased expression in adulthood (23).…”