Calmodulin reverses rundown of L-type Ca2+ channels in guinea pig ventricular myocytes

2004 ◽  
Vol 287 (6) ◽  
pp. C1717-C1724 ◽  
Author(s):  
Jian-Jun Xu ◽  
Li-Ying Hao ◽  
Asako Kameyama ◽  
Masaki Kameyama
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Kinase Inhibitors ◽  
Ventricular Myocytes ◽  
Protein Kinase Inhibitors ◽  
Channel Activity ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Inside Out ◽  
Ca2 Channels

Calmodulin (CaM) is implicated in regulation of Ca2+ channels as a Ca2+ sensor. The effect of CaM on rundown of L-type Ca2+ channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca2+ channel activity disappeared within 1–3 min and did not reappear when the patch was excised and exposed to an artificial intracellular solution. However, application of CaM (0.03, 0.3, 3 μM) + 3 mM ATP to the intracellular solution within 1 min after patch excision resulted in dose-dependent activation of channel activity. Channel activity averaged 11.2%, 94.7%, and 292.9%, respectively, of that in cell-attached mode. Channel activity in inside-out patch mode was induced by CaM + ATP at nanomolar Ca2+ concentrations ([Ca2+]); however, increase to micromolar [Ca2+] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca2+ dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 μM) + ATP induced Ca2+ channel activity to 150%, 100%, 96.9%, 29.3%, and 16.6%, respectively, revealing a time-dependent action of CaM on the channel. CaM added with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) also induced channel activity, although with much lower potency and shorter duration. Protein kinase inhibitors KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide, and K252a (each 1–10 μM) did not block the effect of CaM, indicating that the effect of CaM on the Ca2+ channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca2+ channel activity, showing a cooperative effect of CaM and ATP on the Ca2+ channel. These results suggest that CaM is a crucial regulatory factor of Ca2+ channel basal activity.

PKA and phosphatases attached to the CaV1.2 channel regulate channel activity in cell-free patches

2016 ◽  
Vol 310 (2) ◽  
pp. C136-C141 ◽  
Author(s):  
Jianjun Xu ◽  
Lifeng Yu ◽  
Etsuko Minobe ◽  
Liting Lu ◽  
Ming Lei ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Catalytic Subunit ◽  
Channel Activity ◽  
Activity Recovery ◽  
Dependent Protein ◽  
Excised Patches ◽  
Inside Out

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca2+ channels (CaV1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the CaV1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of CaV1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. CaV1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the CaV1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced CaV1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the CaV1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

scholarly journals ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

1989 ◽  
Vol 94 (4) ◽  
pp. 693-717 ◽  
Author(s):  
B Ribalet ◽  
S Ciani ◽  
G T Eddlestone
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Insulin Secreting Cells ◽  
Dependent Protein ◽  
Inside Out

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase

1997 ◽  
Vol 110 (12) ◽  
pp. 1395-1402 ◽  
Author(s):  
L. Goretzki ◽  
B.M. Mueller
Keyword(s):  
Protein Kinase ◽  
Plasminogen Activator ◽  
Kinase Inhibitors ◽  
Melanoma Cells ◽  
Protein Kinase Inhibitors ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Dependent Protein

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Nitric oxide decreases pacemaker activity in lymphatic vessels of guinea pig mesentery

2001 ◽  
Vol 280 (6) ◽  
pp. H2707-H2716 ◽  
Author(s):  
Pierre-Yves von der Weid ◽  
Jun Zhao ◽  
Dirk F. Van Helden
Keyword(s):  
Nitric Oxide ◽  
Guinea Pig ◽  
Kinase Inhibitors ◽  
Lymphatic Vessels ◽  
Soluble Guanylyl Cyclase ◽  
Protein Kinase Inhibitors ◽  
Pacemaker Activity ◽  
Dependent Protein Kinase ◽  
Microelectrode Recordings ◽  
Dependent Protein

Intracellular microelectrode recordings were used to determine whether nitric oxide (NO), affects the pacemaker events that initiate vasomotion in lymphatic vessels of the guinea pig mesentery. This pacemaker activity is recorded as spontaneous transient depolarizations (STDs) and is likely to arise through synchronized Ca2+ release from intracellular stores. We show here that acetylcholine-induced endothelium-derived NO and exogenous NO released by sodium nitroprusside (SNP; 100 μM) and DEA-NONOate (500 μM) reduced the frequency and amplitude of STDs. This inhibition of STD frequency and amplitude was independent of the NO-induced hyperpolarization of the smooth muscle. The SNP-induced inhibition of STD frequency and amplitude was abolished during superfusion with the soluble guanylyl cyclase inhibitor ODQ (10 μM) and was diminished in the presence of cGMP and cAMP-dependent protein kinase inhibitors. The data are consistent with the hypothesis that NO inhibits vasomotion primarily by production of cGMP and activation of both cGMP- and cAMP-dependent protein kinases, which reduce the size and frequency of STDs, probably by acting on the underlying synchronized Ca2+ release from intracellular stores.

scholarly journals Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes†

2009 ◽  
Vol 85 (3) ◽  
pp. 454-463 ◽  
Author(s):  
Takeshi Aiba ◽  
Geoffrey G. Hesketh ◽  
Ting Liu ◽  
Rachael Carlisle ◽  
Maria Celeste Villa-Abrille ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Na Channel ◽  
Dependent Protein Kinase ◽  
Channel Regulation ◽  
Protein Kinase Ii ◽  
Dependent Protein

scholarly journals Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

2015 ◽  
Vol 36 (2) ◽  
pp. 555-568 ◽  
Author(s):  
Jiaoqian Ying ◽  
Yuan Zhang ◽  
Shan Gong ◽  
Zhigang Chang ◽  
Xiaofeng Zhou ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Dependent Manner ◽  
The Novel ◽  
Inotropic Effects ◽  
Kinase C ◽  
Melanocortin 4 Receptor ◽  
Dose Dependent ◽  
Ca2 Channels

Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca2+ currents (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased ICa,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-β-S or anti-Gβ antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca2+-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKCθ), and PKCθ inhibition abolished the decrease in ICa,L induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca2+ channels via the MC4-R that couples sequentially to the βγ subunits of Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes.

Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes

1994 ◽  
Vol 267 (2) ◽  
pp. H812-H820 ◽  
Author(s):  
A. Mattiazzi ◽  
L. Hove-Madsen ◽  
D. M. Bers
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Inhibitory Effect ◽  
Ruthenium Red ◽  
Protein Kinase Inhibitors ◽  
Inhibitory Peptide ◽  
Dependent Protein Kinase ◽  
Ca Transport ◽  
Ca Uptake ◽  
Dependent Protein

Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca-adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin-permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 microM ruthenium red and 10 mM oxalate. neither N5,2'-w-dibutyryl-cAMP (up to 500 microM) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 microM) affected the maximum uptake rate (Vmax) or the dissociation constant (Kd) for Ca uptake. However, the PKA inhibitor H-89 significantly increased Kd (e.g., from 307 +/- 67 to 826 +/- 62 nM Ca at 40-65 microM H-89) without significantly affecting Vmax. Both CaM-KII inhibitors, KN-62 (60 microM) and a CaM-KII inhibitory peptide (10 microM), significantly decreased Vmax from 11.95 +/- 0.5 to 9.48 +/- 0.6 nmol.mg-1.min-1 and from 10.95 +/- 1.72 to 7.37 +/- 0.94 nmol.mg-1.min-1, respectively, without consistently changing Kd. The effects of H-89 on Kd and of KN-62 on Vmax were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca-ATPase).(ABSTRACT TRUNCATED AT 250 WORDS)

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