ACh‐induced rebound stimulation of L‐type Ca²⁺ current in guinea‐pig ventricular myocytes, mediated by Gβγ‐dependent activation of adenylyl cyclase

Abstract

The effects that muscarinic receptor stimulation have on the cAMP‐dependent regulation of L‐type Ca2+ currents were studied in isolated guinea‐pig ventricular myocytes using the whole‐cell configuration of the patch‐clamp technique. The muscarinic agonist ACh inhibited the Ca2+ current stimulated by the β‐adrenergic agonist isoprenaline (Iso), and washout of ACh revealed a stimulatory response that appeared as a transient rebound increase in the amplitude of the Ca2+ current. The ACh‐induced stimulatory effect was not observed in the absence of Iso. ACh‐induced rebound stimulation was also observed in the presence of H2 histamine receptor activation and cholera toxin treatment, which like β‐adrenergic receptor activation enhance adenylyl cyclase (AC) activity in a stimulatory G protein (Gs)‐dependent manner. ACh‐induced rebound stimulation was not observed in the presence of forskolin, which enhances AC activity in a Gs‐independent manner. Pertussis toxin (PTX) treatment blocked both the stimulatory and inhibitory effects of ACh. Intracellular dialysis with QEHA, a peptide that binds free G protein βγ subunits, selectively antagonized the stimulatory effect, leaving an enhanced inhibitory effect. Evidence for the expression of AC4, an isoform of AC that can be stimulated by Gβγ but only in the presence of Gαs, was obtained by Western blot analysis of guinea‐pig ventricular myocyte membrane preparations. These results suggest that muscarinic receptor stimulation facilitates as well as inhibits cAMP‐dependent regulation of the Ca2+ current and that the net response is a balance between these two actions. We suggest that the stimulatory effect is due to a direct activation of AC4 by the βγ subunits of a PTX‐sensitive G protein.

Adenylyl cyclases 5 and 6 underlie PIP3-dependent regulation

“…3C), as described previously for cardiac myocytes (14,37). Therefore, it can be concluded that G i/o proteins mediate this effect.…”

“…To measure real-time dynamics of the well-described cAMP rebound phenomenon after withdrawal of muscarinic stimulation (14,16,37), we chose atrial myocytes, because overall they show more responsiveness to muscarinic receptor stimulation than do ventricular myocytes (38). We therefore acutely isolated atrial myocytes from transgenic mice expressing the well-established cAMP biosensor Epac1-camps (35) and imaged cAMP levels by means of FRET.…”

“…Those isoforms are extremely sensitive toward activation of G i/o -coupled receptors, most likely because of their prolonged interactions with Ga subunits (13). In addition to the direct inhibition of cAMP responses via inhibitory G (G i ) proteins, a second level of cAMP regulation via G i proteins has been reported (14)(15)(16), for which the mechanism is not yet clear. In cardiac myocytes, most prominent in atrial myocytes, it has been observed that M 2 AChR activation not only inhibits b-adrenergic responses, it also facilitates b-adrenergic responses immediately after the termination of M 2 AChR stimulation (defined as a rebound response) (14)(15)(16)(17)(18).…”

“…In addition to the direct inhibition of cAMP responses via inhibitory G (G i ) proteins, a second level of cAMP regulation via G i proteins has been reported (14)(15)(16), for which the mechanism is not yet clear. In cardiac myocytes, most prominent in atrial myocytes, it has been observed that M 2 AChR activation not only inhibits b-adrenergic responses, it also facilitates b-adrenergic responses immediately after the termination of M 2 AChR stimulation (defined as a rebound response) (14)(15)(16)(17)(18). These rebound effects can explain physiologic phenomena such as postvagal tachycardia (19) and arrhythmogenic mechanisms (20).…”

Characterization of Ca2+Channels and G Proteins Involved in Arachidonic Acid Release by Endothelin-1/EndothelinAReceptor

“…Cholera toxin activates G S via a receptor-independent mechanism (Belevych et al, 2001). Treatment with 1 g/ml cholera toxin failed to induce AA release (Fig.…”