Transfection of a phosphatidyl-4-phosphate 5-kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and alpha-adrenergic agonists

GIRK (G protein-activated inward-rectifying K(+) channel) channels, important regulators of membrane excitability in the heart and in the central nervous, are activated by interaction with betagamma subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P(2) levels relevant to GIRK channel regulation are under regulatory control of receptors activating phospholipase C. In the present study a phosphatidyl-4-phosphate 5-kinase was expressed in atrial myocytes by transient transfection. This did not affect basal properties of GIRK current activated by acetylcholine via M(2) receptors but completely abolished inhibition of guanosine triphosphate-gamma-S activated current by endothelin-1 or alpha-adrenergic agonists. We conclude that though PtIns(4,5)P(2) is conditional for channel gating, its normal level in the membrane is not limiting basal function of GIRK channels. Moreover, our data provide further evidence for a regulation of GIRK channels by alpha(1A) receptors and endothelin-A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P(2).     

Overexpression of monomeric and multimeric GIRK4 subunits in rat atrial myocytes removes fast desensitization and reduces inward rectification of muscarinic K(+) current (I(K(ACh))). Evidence for functional homomeric GIRK4 channels

K(+) channels composed of G-protein-coupled inwardly rectifying K(+) channel (GIRK) (Kir3.0) subunits are expressed in cardiac, neuronal, and various endocrine tissues. They are involved in inhibiting excitability and contribute to regulating important physiological functions such as cardiac frequency and secretion of hormones. The functional cardiac (K((ACh))) channel activated by G(i)/G(o)-coupled receptors such as muscarinic M(2) or purinergic A(1) receptors is supposed to be composed of the subunits GIRK1 and GIRK4 in a heterotetrameric (2:2) fashion. In the present study, we have manipulated the subunit composition of the K((ACh)) channels in cultured atrial myocytes from hearts of adult rats by transient transfection of vectors encoding for GIRK1 or GIRK4 subunits or GIRK4 concatemeric constructs and investigated the effects on properties of macroscopic I(K(ACh)). Transfection with a GIRK1 vector did not cause any measurable effect on properties of I(K(ACh)), whereas transfection with a GIRK4 vector resulted in a complete loss in desensitization, a reduction of inward rectification, and a slowing of activation. Transfection of myocytes with a construct encoding for a concatemeric GIRK4(2) subunit had similar effects on desensitization and inward rectification. Following transfection of a tetrameric construct (GIRK4(4)), these changes in properties of I(K(ACh)) were still observed but were less pronounced. Heterologous expression in Chinese hamster ovary cells and human embryonic kidney 293 cells of monomeric, dimeric, and tetrameric GIRK4 resulted in robust currents activated by co-expressed A(1) and M(2) receptors, respectively. These data provide strong evidence that homomeric GIRK4 complexes form functional G(beta)gamma gated ion channels and that kinetic properties of GIRK channels, such as activation rate, desensitization, and inward rectification, depend on subunit composition.     

Role of internalization of M2 muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+ current in heart

In the heart, ACh activates the ACh-activated K(+) current (I(K,ACh)) via the M(2) muscarinic receptor. The relationship between desensitization of I(K,ACh) and internalization of the M(2) receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, I(K,ACh) declined by approximately 62% with time constants of 1.5 and 26.9 min, whereas approximately 83% of the M(2) receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with beta-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and beta-arrestin 2 significantly increased I(K,ACh) desensitization and M(2) receptor internalization during a 3-min application of agonist. Internalized M(2) receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and beta-arrestin 2-dependent internalization of the M(2) receptor into clathrin-coated vesicles could play a major role in I(K,ACh) desensitization.     

Phosphatidylinositol 4,5-bisphosphate is acting as a signal molecule in alpha(1)-adrenergic pathway via the modulation of acetylcholine-activated K(+) channels in mouse atrial myocytes

We have investigated the effect of alpha(1)-adrenergic agonist phenylephrine (PE) on acetylcholine-activated K(+) currents (I(KACh)). I(KACh) was recorded in mouse atrial myocytes using the patch clamp technique. I(KACh) was activated by 10 microm ACh and the current decreased by 44.27 +/- 2.38% (n = 12) during 4 min due to ACh-induced desensitization. When PE was applied with ACh, the extent of desensitization was markedly increased to 69.34 +/- 2.22% (n = 9), indicating the presence of PE-induced desensitization. I(KACh) was fully recovered from desensitization after a 6-min washout. PE-induced desensitization of I(KACh) was not affected by protein kinase C inhibitor, calphostin C, but abolished by phospholipase C (PLC) inhibitor, neomycin. When phophatidylinositol 4,5-bisphosphate (PIP(2)) replenishment was blocked by wortmannin (an inhibitor of phophatidylinositol 3-kinase and phophatidylinositol 4-kinase), desensitization of I(KACh) in the presence of PE was further increased (97.25 +/- 7.63%, n = 6). Furthermore, the recovery from PE-induced desensitization was inhibited, and the amplitude of I(KACh) at the second exposure after washout was reduced to 19.65 +/- 2.61% (n = 6) of the preceding level. These data suggest that the K(ACh) channel is modulated by PE through PLC stimulation and depletion of PIP(2).     

Hypercholesterolemia induces up-regulation of KACh cardiac currents via a mechanism independent of phosphatidylinositol 4,5-bisphosphate and Gβγ

Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of K(ACh) mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on K(ACh) is unknown. Here we show that cholesterol plays a critical role in modulating K(ACh) currents (I(K,ACh)) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed I(K,ACh). Moreover, a high-cholesterol diet resulted in up to 3-fold increase in I(K,ACh) in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies I(K,ACh). Furthermore, our data suggest that cholesterol affects I(K,ACh) via a mechanism which is independent of both PI(4,5)P(2) and Gβγ. Interestingly, the effect of cholesterol on I(K,ACh) is opposite to its effect on I(K1) in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating I(K,ACh) in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.     

Combined phosphoinositide and Ca2+ signals mediating receptor specificity toward neuronal Ca2+ channels

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates Ca(2+) (I(Ca)) and M-type K(+) currents in superior cervical ganglion sympathetic neurons. In those cells, M(1) muscarinic and AT(1) angiotensin types do not elicit Ca(2+)(i) signals and suppress both currents via depletion of PIP(2), whereas the B(2) bradykinin and P2Y purinergic types elicit robust IP(3)-mediated [Ca(2+)](i) rises and neither deplete PIP(2) nor inhibit I(Ca). We have suggested that this specificity arises from differential Ca(2+)(i) signals underlying receptor-specific stimulation of PIP(2) synthesis by phosphatidylinositol (PI) 4-kinase. Here, we investigate which PI 4-kinase isoform underlies this signal, whether stimulation of PI 4-phosphate 5-kinase is also required, and the origin of receptor-specific Ca(2+)(i) signals. Recordings of I(Ca) were used as a PIP(2) "biosensor." In control, stimulation of M(1), but not B(2) or P2Y, receptors robustly suppressed I(Ca). However, when PI 4-kinase IIIβ, diacylglycerol kinase, Rho, or Rho-kinase was blocked, agonists of all three receptors robustly suppressed I(Ca). Overexpression of exogenous M(1) receptors yielded large [Ca(2+)](i) rises by muscarinic agonist, and transfection of wild-type IRBIT decreased Ca(2+)(i) signals, whereas dominant negative IRBIT-S68A had little effect on B(2) or P2Y responses but greatly increased muscarinic responses. We conclude that overlaid on microdomain organization is IRBIT, setting a "threshold" for [IP(3)], assisting in fidelity of receptor specificity.     

Autonomic regulation of calcium and potassium channels is oppositely modulated by microtubules in cardiac myocytes

We recently showed that colchicine treatment of rat ventricular myocytes increases the L-type Ca2+ current (I(Ca)) and intracellular Ca2+ concentration ([Ca2+](i)) transients and interferes with adrenergic signaling. These actions were ascribed to adenylyl cyclase (AC) stimulation after G(s) activation by alpha,beta-tubulin. Colchicine depolymerizes microtubules into alpha,beta-tubulin dimers. This study analyzed muscarinic signals in myocytes with intact or depolymerized microtubules. Myocytes were loaded with the Ca2+ indicator fluo 3 and were field stimulated at 1 Hz or voltage clamped. In untreated cells, carbachol (CCh; 1 microM) induced ACh-activated K(+) current [I(K(ACh))], which happens via betagamma-subunits from the activation of G(i). Carbachol also reduced [Ca2+](i) transients and contractions. Once G(i) is activated by muscarinic agonist, the alpha(i)-subunit is released from the betagamma-subunits, but it is silent, and its inhibition of the AC/cAMP cascade, manifested by I(Ca) reduction, is not seen unless AC has been previously activated. In colchicine-treated cells, CCh caused greater reductions of [Ca2+](i) transients and contractions than in untreated cells. The alpha(i)-subunit became effective in signaling through the AC/cAMP cascade and reduced I(Ca) without changing its voltage-dependence. Isoproterenol (Iso) regained its efficacy and reversed I(Ca) inhibition by CCh. Stimulation of I(Ca) by forskolin persisted in colchicine-treated cells when Iso was ineffective. The effect of CCh on I(K(ACh)) was occluded in colchicine-treated cells. Colchicine treatment, per se, may increase I(K(ACh)) by betagamma-subunits released from G(s) to mask this effect of CCh. Microtubules suppress I(Ca) regulation by alpha(i); their disruption releases restraints that unmask muscarinic inhibition of I(Ca). Summarily, colchicine treatment reverses regulation of ventricular excitation-contraction coupling by autonomic agents.     

Coupling to Gs and G(q/11) of histamine H2 receptors heterologously expressed in adult rat atrial myocytes

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.     

Comparative research on synaptic transmission in the sympathetic ganglia of rabbits and frogs during acetylcholinesterase inhibition

Organophosphorus inhibitor of acetylcholinesterase (AChE) armin (1 x 10(-6) M) induced a variety of pre- and postsynaptic effects resulting from the AChE inhibition and subsequent accumulation of acetylcholine (ACh) in the synaptic cleft. The intensity of postsynaptic effects (level of neuron depolarization, degree of action potential depression) was shown to be different in the ganglia of frog and rabbit. This could be explained by differences in the total amount of ACh released in response to nerve stimulation as well as at rest. Both muscarinic and nicotinic cholinoreceptors were involved in the process of sustained depolarization of the neurons in the rabbit superior cervical ganglion after AChE inhibition. In frog ganglion neurons the nicotinic receptors did not participate in depolarization evidently due to their fast desensitization. The activation of presynaptic muscarinic receptors resulted in decrease of ACh released by nerve stimulation seems to weaken depolarization and blockade of synaptic transmission in sympathetic ganglia treated by AChE inhibitors.     

Novel inhibition of gbetagamma-activated potassium currents induced by M(2) muscarinic receptors via a pertussis toxin-insensitive pathway

G(i) protein-coupled receptors such as the M(2) muscarinic acetylcholine receptor (mAChR) and A(1) adenosine receptor have been shown to activate G protein-activated inwardly rectifying K(+) channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M(2) receptors not only activate but also reversibly inhibit these K(+) currents when stimulated with agonist for up to 2 min. The M(2) mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5'-O-(thiotriphosphate) in the pipette. Under these conditions the M(2) mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gbetagamma, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gbetagamma subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M(2) mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M(2) mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M(4) mAChR and alpha(1A) adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A(1) and A(3) adenosine receptors and alpha(2A) and alpha(2C) adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M(2) and M(4) mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gbetagamma-activated GIRK currents in a membrane-delimited fashion.     

麻醉剂氟烷对心脏毒蕈碱型钾通道的影响

神经递质乙酰胆碱（ＡＣｈ）调节心脏功能最重要的离子通道就暗毒蕈碱型钾通道（ｉＫ,ＡＣｈ）,该通道由ＡＣｈ经鸟苷酸调节蛋白（Ｇ蛋白）的βγ亚单位而激活。本实验彩全细胞膜片箝方法,观察了麻醉药氟烷对豚鼠心房肌细胞ｉＫ,ＡＣｈ的影响。氟烷对ｉＫ,ＡＣｈ电流具抑制效应,灌注之后可使ＡＣｈ激活的ｉＫ,ＡＣｈ速率减慢,峰植下降。但其抑制ｉＫ,ＡＣｈ的程度依激活方式而异：经正常激活途径,即由ＡＣｈ激活毒蕈碱Ｍ样     

Control of the cardiac muscarinic K+ channel by beta-arrestin 2

Control of the cardiac muscarinic K(+) current (i(K,ACh)) by beta-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K(+) channel, receptor kinase (GRK2), and beta-arrestin 2, desensitization of i(K,ACh) during a 3-min application of 10 micrometer ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of beta-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of i(K,ACh) in cells transfected with beta-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of i(K,ACh) on application and wash-off of ACh in cells transfected with beta-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak i(K,ACh)) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active beta-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 +/- 0.05 to 0.1 +/- 0.01 nA). We conclude that beta-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K(+) channel.     

Activation of muscarinic receptors reduces store-operated Ca2+ entry in HEK293 cells

In many cell types membrane receptors for hormones or neurotransmitters activate a signal transduction pathway which releases Ca2+ from intracellular Ca2+ stores by the second messenger inositol 1,4,5-trisphosphate. As a consequence store-operated Ca2+ entry (SOCE) becomes activated. In the present study we addressed the question if receptor/agonist binding can modulate Ca2+ entry by mechanisms different from the store-operated one. Therefore SOCE was examined in HEK293 cells microscopically with the fura-2 technique and with patch clamp. We found that maximally preactivated SOCE could, concentration dependently, be reduced up to 80% by the muscarinic agonist acetylcholine when the cytoplasmic Ca2+ concentration was used as a measure. Muscarinic receptors seem to mediate this decrease since atropine blocked the effect completely and cell types without muscarinic receptors (BHK21, CHO) did not show acetylcholine-induced decrease of Ca2+ entry. Moreover expression of muscarinic receptor subtypes M1 and M3 in BHK21 cells established the muscarinic decrease of SOCE. Electrical measurements revealed that the membrane potential of HEK293 cells did not show any response to ACh, excluding that changes of driving forces are responsible for the block of Ca2+ entry. In contrast the electrical current which is responsible for SOCE in HEK293 cells (Ca2+ release-activated Ca2+ current (I(CRAC)) was inhibited (maximally 55%) by 10 microM ACh. From these data we conclude that in HEK293 cells a muscarinic signal transduction pathway exists which decreases the cytoplasmic Ca2+ concentration by an inhibition of I(CRAC). This mechanism may serve as a modulator of Ca2+ entry preventing a Ca2+ overload of the cytoplasm after Ca2+ store depletion.     

Species- and tissue-dependent effects of NO and cyclic GMP on cardiac ion channels

Biochemical studies have established the presence of a NO pathway in the heart, including sources of NO and various effectors. Several cardiac ion channels have been shown to be modified by NO, such as L-type Ca(2+), ATP-sensitive K(+), and pacemaker f-channels. Some of these effects are mediated by cGMP, through the activity of three main proteins: the cGMP-dependent protein kinase (PKG), the cGMP-stimulated phosphodiesterase (PDE2) and the cGMP-inhibited PDE (PDE3). Other effects appear independent of cGMP, as for instance the NO modulation of the ryanodine receptor-Ca(2+) channel. In the case of the cardiac L-type Ca(2+) channel current (I(Ca,L)), both cGMP-dependent and cGMP-independent effects have been reported, with important tissue and species specificity. For instance, in rabbit sinoatrial myocytes, NO inhibits the beta-adrenergic stimulation of I(Ca,L) through activation of PDE2. In cat and human atrial myocytes, NO potentiates the cAMP-dependent stimulation of I(Ca,L) through inhibition of PDE3. In rabbit atrial myocytes, NO enhances I(Ca,L) in a cAMP-independent manner through the activation of PKG. In ventricular myocytes, NO exerts opposite effects on I(Ca,L): an inhibition mediated by PKG in mammalian myocytes but by PDE2 in frog myocytes; a stimulation attributed to PDE3 inhibition in frog ventricular myocytes but to a direct effect of NO in ferret ventricular myocytes. Finally, NO can also regulate cardiac ion channels by a direct action on G-proteins and adenylyl cyclase.     

Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes

Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K(+) current (I(K(ACh))) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by approximately 25% (vs. untreated controls) in field-stimulated myocytes. S1P(1) was shown to be involved by using the S1P(1)-selective agonist SEW2871 on myocytes isolated from S1P(3)-null mice. However, in these myocytes, S1P(3) can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P(1) antagonist VPC23019. Since S1P(1) activates G(i) exclusively, whereas S1P(3) activates both G(i) and G(q), these results strongly implicate the involvement of mainly G(i). Additional experiments using the I(K(ACh)) blocker tertiapin demonstrated that I(K(ACh)) can contribute to the negative inotropy following S1P activation of S1P(1) (perhaps through G(ibetagamma) subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I(K(ACh))) can reduce L-type calcium current and thus can decrease the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G(i) to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca(2+)](i); and 2) a second pathway that acts via G(i) to activate I(K(ACh)) and reduce APD. This decrease in APD is expected to decrease Ca(2+) influx and reduce [Ca(2+)](i) and myocyte contractility.     

Molecular mechanism of doxorubicin-induced anticholinergic effect in guinea-pig atria

The molecular mechanisms of anticholinergic actions of doxorubicin were examined by electrophysiological methods in atria and myocytes isolated from guinea-pig heart. A direct anticholinergic action of doxorubicin was confirmed with antagonistic action on carbachol-induced negative inotropic effect in atria. Both carbachol and adenosine produced shortening of action potential duration in atria measured by a microelectrode method. Doxorubicin (10-100 microM) inhibited the carbachol-induced action potential shortening in a concentration-dependent manner. However, doxorubicin did not antagonize the shortening elicited by adenosine. The whole-cell voltage clamp technique was performed to induce the muscarinic acetylcholine-receptor-operated K+ current (IK.ACh) in atrial myocytes loaded with GTP or GTPgammaS, a nonhydrolysable analogue of GTP. Doxorubicin (1-100 microM) suppressed carbachol-induced IK.ACh in a concentration-dependent manner (IC50 = 5.6 microM). In contrast, doxorubicin (10 and 100 microM) suppressed neither adenosine-induced IK.ACh nor GTPgammaS-induced IK.ACh. These results indicate that doxorubicin produces a direct anticholinergic effect through the muscarinic receptors in atrial myocytes.     

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of I(to) and augmentation of K(ATP) channels

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.     

Acetylcholine and the myocardium: electrophysiological aspects

The cardiac inhibitory effects (negative inotropic and chronotropic) of muscarinic cholinergic stimulation by acetylcholine (ACh) are well established. They are due to electrophysiological modifications involving (1) the activation of the resting K+ channel showing inward going rectification properties; (2) the reduction of the inward calcium current (I Ca). Recent works on isolated myocardial cells allowed to investigate the molecular mechanisms involved between muscarinic cholinergic receptors activation and effector (the ionic channel). The results indicate that muscarinic receptor communicates with the K+ channel, via GTP-binding protein (Ni, o or G) and that does not involve adenylate-cyclase. In contrast to the direct muscarinic activation of K+ channel, ACh decreases I Ca by inhibiting, via Ni, the cAMP production. The inhibition of I Ca is larger in the beta-stimulated cells.     

Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits 'fade' or desensitization in the AVN, as established for the heart's primary pacemaker--the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1-1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at -120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at -120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.