Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.     

Acetylcholine increases intracellular Ca2+ in the rat pituitary folliculostellate cells in primary culture

Pituitary folliculostellate cells (FSCs) are thought to partially inhibit pituitary hormone secretion through a paracrine mechanism. In this process, one of the important questions is what factors regulate the function of FSCs. Because ACh is synthesized in and possibly released from the corticotrophs and lactotrophs, we examined whether FSCs respond to ACh by the method of Ca2+ imaging in primary cultured FSCs from male Wistar rats. ACh (30 nM-3 microM) increased intracellular calcium concentration ([Ca2+](i)) of FSCs in a concentration-dependent manner, with an initial rapid rise followed by a relatively sustained increase. The complete block of the response by atropine and pirenzepine suggests involvement of muscarinic receptors. Depletion of the stored Ca2+ by thapsigargin blocked the response completely. Blockers of phospholipase C, U-73122 and neomycin, suppressed significantly the rise of [Ca2+](i). These results suggest that ACh increases [Ca2+](i) in FSCs by activating phospholipase C, presumably through activation of M(1) receptors. The rise in [Ca2+](i) could trigger a variety of Ca2+-dependent cellular processes, including the synthesis and release of bioactive substances, which in turn act on endocrine cells.     

Changes in functioning of rat submandibular salivary gland under streptozotocin-induced diabetes are associated with alterations of Ca2+ signaling and Ca2+ transporting pumps

Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca2+ indicators fura-2/AM for measuring cytosolic Ca2+ concentration ([Ca2+]i) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca2+]i and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca2+]i transients in acinar cells from diabetic animals. However, [Ca2+]i transients mediated by Ca2+ release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca2+-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca2+ release from ER under the diabetes. Both plasmalemma and ER Ca2+-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca2+ overload, increased Ca2+-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca2+-ATPases activity and ER Ca2+ content.     

The sodium pump modulates the influence of I(Na) on [Ca2+]i transients in mouse ventricular myocytes

To investigate whether activity of the sarcolemmal Na pump modulates the influence of sodium current on excitation-contraction (E-C) coupling, we measured [Ca(2+)](i) transients (fluo-3) in single voltage-clamped mouse ventricular myocytes ([Na+](pip) = 15 or 0 mM) when the Na pump was activated (4.4 mM K(+)(o)) and during abrupt inhibition of the pump by exposure to 0 K with a rapid solution-switcher device. After induction of steady state [Ca2+](i) transients by conditioning voltage pulses (0.25 Hz), inhibition of the Na pump for 1.5 s immediately before and continuing during a voltage pulse (200 ms, -80 to 0 mV) caused a significant increase (15 +/- 2%; n = 16; p < 0.01) in peak systolic [Ca2+](i) when [Na+](pip) was 15 mM. In the absence of sodium current (I(Na), which was blocked by 60 microM tetrodotoxin (TTX)), inhibition of the Na pump immediately before and during a voltage pulse did not result in an increase in peak systolic [Ca2+](i). Abrupt blockade of I(Na) during a single test pulse with TTX caused a slight decrease in peak [Ca2+](i), whether the pump was active (9%) or inhibited (10%). With the reverse-mode Na/Ca exchange inhibited by KB-R 7943, inhibition of the Na pump failed to increase the magnitude of the peak systolic [Ca2+](i) (4 +/- 1%; p = NS) when [Na+](pip) was 15 mM. When [Na+](pip) was 0 mM, the amplitude of the peak systolic [Ca2+](i) was not altered by abrupt inhibition of the Na pump immediately before and during a voltage pulse. These findings in adult mouse ventricular myocytes indicate the Na pump can modulate the influence of I(Na) on E-C coupling in a single beat and provide additional evidence for the existence of Na fuzzy space, where [Na+] can significantly modulate Ca2+ influx via reverse Na/Ca exchange.     

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     

Roles of mitochondria and temperature in the control of intracellular calcium in adult rat sensory neurons

We recorded Ca2+ current and intracellular Ca2+ ([Ca2+](i)) in isolated adult rat dorsal root ganglion (DRG) neurons at 20 and 30 degrees C. In neurons bathed in tetraethylammonium and dialyzed with cesium, warming reduced resting [Ca2+](i) from 87 to 49 nM and the time constant of the decay of [Ca2+](i) transients (tau(r)) from 1.3 to 0.99s (Q(10)=1.4). The Buffer Index, the ratio between Ca2+ influx and Delta[Ca2+](i) (f I(ca)d(t)/Delta[Ca2+]i) , increased two- to threefold with warming. Neither inhibition of the plasma membrane Ca2+ -ATPase by intracellular sodium orthovanadate nor inhibition of Ca2+ uptake by the endoplasmic reticulum by thapsigargin plus ryanodine were necessary for the effects of warming on these parameters. In contrast, inhibition of the mitochondrial Ca2+ uniporter by intracellular ruthenium red largely reversed the effects of warming. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 500 nM) increased resting [Ca2+](i) at 30 degrees C. Ten millimolar intracellular sodium prolonged the recovery of [Ca2+](i) transients to 10-40s. This effect was reversed by an inhibitor of mitochondrial Na(+)/Ca2+ -exchange (CGP 37157, 10 microM). Thus, mitochondrial Ca2+ uptake is necessary for the temperature-dependent increase in Ca2+ buffering and mitochondrial Ca2+ fluxes contribute to the control of [Ca2+](i) between 50 and 150 nM at 30 degrees C.     

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.     

氨甲酰胆碱通过M2毒蕈碱受体增加豚鼠心肌细胞钠钙交换电流

本文旨在研究氨甲酰胆碱(carbachol, CCh)对豚鼠心肌的正性变力性机制。用Axon200A膜片钳放大器观察CCh 对电压钳制下的豚鼠心肌细胞L-型钙电流(ICa)和钠钙交换电流(INa/Ca)的效应。结果表明, CCh(100 μmol/L)分别使正向INa/Ca从对照组的(1.2 ± 0.1) pA/pF 增加到(2.0 ± 0.3) pA/pF,使反向 INa/Ca 从对照组的(1.3 ± 0.5) pA/pF 增加到(2.1 ± 0.8) pA/pF (P<0.01)。CCh对ICa无影响。CCh 对INa/Ca的激动作用可被阿托品和methoctramine所阻断。以上结果提示, CCh 对豚鼠心脏的正性变力作用是通过激动了钠钙交换,而且是 M2 毒蕈碱受体所介导的。     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

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.     

Beta-angonists modulate ACh-inhibition of a K current in intestinal smooth muscle cells.

ACh causes a long-lasting inhibition of STOCs via G proteins in intestinal smooth muscle cells. We examined the effects of isoproterenol (Iso) on the ACh-induced inhibition of STOCs in isolated ileal smooth muscle cells using the G omega-seal whole cell clamp technique. In control, ACh (1 microM) completely suppressed STOCs, which did not desensitize over a period lasting 20 minutes. When Iso (10 microM) was added to the bath in the presence of ACh, the ACh-induced inhibition of STOCs was gradually removed. This effect of Iso was prevented by propranolol (10 microM). Application of Db-cAMP (500 microM) mimicked the Iso effects. Intracellulary applied GTP-gamma S (100 microM) gradually suppressed STOCs in the absence of ACh, which could not be removed by either Iso or Db-cAMP. These results suggest that beta-adrenergic stimulation causes a removal of the muscarinic inhibition of STOCs via a cAMP-dependent process.     

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.     

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.     

Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors

We investigated the functional properties of rat alpha9 and alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfection in the rat GH4C1 cell line, using both Ca(2+) imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca(2+) transients, suppressed in Ca(2+)-free medium and invariably accompanied by the activation of whole-cell inward currents. The mean amplitude of ACh-induced currents was as small as -16 pA in alpha9 subunit cDNA-transfected GH4C1 cells (alpha9-GH4C1), while they were much larger (range: -150 to -300 pA) in alpha9alpha10 subunit cDNAs-transfected GH4C1 cells (alpha9alpha10-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca(2+) permeability of alpha9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike alpha9- and alpha9alpha10-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca(2+) transients, the latter being detected in the absence of Ca(2+) in the extracellular medium and being suppressed by the Ca(2+)-ATPase inhibitor thapsigargin, known to deplete IP(3)-sensitive stores. These results indicated the involvement of muscarinic nAChRs and the lack of functional ACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca(2+) current (P(f), i.e. the percentage of total current carried by Ca(2+) ions) in alpha9alpha10-GH4C1, obtaining a P(f) value of 22 +/- 4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca(2+) entry through alpha9-containing nAChRs gated by ACh is discussed.     

A cellular mechanism for nitric oxide-mediated cholinergic control of mammalian heart rate

The biochemical signaling pathways involved in nitric oxide (NO)- mediated cholinergic inhibition of L-type Ca2+ current (ICa[L]) were investigated in isolated primary pacemaker cells from the rabbit sinoatrial node (SAN) using the nystatin-perforated whole-cell voltage clamp technique. Carbamylcholine (CCh; 1 microM), a stable analogue of acetylcholine, significantly inhibited ICa(L) after it had been augmented by isoproterenol (ISO; 1 microM). CCh also activated an outward K+ current, IK(ACh). Both of these effects of CCh were blocked completely by atropine. Preincubation of the SAN cells with L-nitro- arginine methyl ester (L-NAME; 0.2-1 mM), which inhibits NO synthase (NOS), abolished the CCh-induced attenuation of ICa(L) but had no effect on IK(ACh). Coincubation of cells with both L-NAME and the endogenous substrate of NOS, L-arginine (1 nM), restored the CCh- induced attenuation of ICa(L), indicating that L-NAME did not directly interfere with the muscarinic action of CCh on ICa(L). In the presence of ISO the CCh-induced inhibition of ICa(L) could be mimicked by the NO donor 3-morpholino-sydnonimine (SIN-1; 0.1 mM). SIN-1 had no effect on its own or after a maximal effect of CCh had developed, indicating that it does not inhibit ICa(L) directly. SIN-1 failed to activate IK(ACh), demonstrating that it did not activate muscarinic receptors. Both CCh and NO are known to activate guanylyl cyclase and elevate intracellular cGMP. External application of methylene blue (10 microM), which interferes with the ability of NO to activate guanylyl cyclase, blocked the CCh-induced attenuation of ICa(L). However, it also blocked the activation of IK(ACh), suggesting an additional effect on muscarinic receptors or G proteins. To address this, a separate series of experiments was performed using conventional whole-cell recordings with methylene blue in the pipette. Under these conditions, the CCh-induced attenuation of ICa(L) was blocked, but the activation of IK(ACh) was still observed. Methylene blue also blocked the SIN-1-induced decrease in ICa(L). 6-anilino-5,8-quinolinedione (LY83583; 30 microM), an agent known to decrease both basal and CCh-stimulated cGMP levels, prevented the inhibitory effects of both CCh and SIN-1 on ICa(L), but had no effect on the activation of IK(ACh) by CCh. In combination, these results show that CCh- and NO-induced inhibition of ICa(L) is mediated by cGMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pulmonary vascular response to normoxia and K(Ca) channel activity is developmentally regulated

To address developmental regulation of pulmonary vascular O(2) sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O(2), 2) Ca2+-sensitive K(+) (K(Ca)) channel activity decreases with maturation, and 3) PASMC K(Ca) channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O(2) tension. Acute normoxia induced a 22 +/- 2% decrease in cytosolic Ca2+ concentration ([Ca2+](i)) in fetal PASMCs and no change in ([Ca2+](i)) in adult PASMCs (P < 0.01). The effects of K(+) channel antagonists were studied on fetal and adult PASMC ([Ca2+](i)). Iberiotoxin (10(-9) M) caused PASMC ([Ca2+](i)) to increase by 694 +/- 22% in the fetus and caused no change in adult PASMCs. K(Ca) channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both K(Ca) channel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O(2) sensing render the fetal PASMCs uniquely sensitive to an acute increase in O(2) tension at a biologically critical time point.     

Acetylcholine-induced calcium signalling in adrenaline- and noradrenaline-containing adrenal chromaffin cells

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group (n-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group (m-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on n- and m-type cells, respectively. Cytochemical staining showed that n-cells contained adrenaline, whereas m-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.     

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.     

Regulation by clozapine of calcium handling by rat submandibular acinar cells

The effect of clozapine on the intracellular concentration of calcium ([Ca2+](i)) in rat submandibular acinar cells was tested. By itself clozapine had no effect on the mobilization of intracellular pools of calcium or on the uptake of extracellular calcium. It inhibited the increase of the [Ca2+](i) in response to carbachol (half-maximal inhibitory concentrations, IC(50)=100nM) and to norepinephrine and epinephrine (IC(50)=10nM) without affecting the response to substance P, extracellular ATP or thapsigargin. Clozapine inhibited the uptake of extracellular calcium in response to epinephrine but not to substance P, ATP or thapsigargin. It also decreased the production of inositol phosphates elicited by epinephrine but not by substance P or fluoride. It is concluded that, by itself, clozapine has no effect on the [Ca2+](i) in rat salivary acinar cells. It selectively inhibits muscarinic and adrenergic receptors in the acinar plasma membrane.