Modulation of extracellular ATP-induced Ca2+ responses: role of protein kinases

Evidence for the modulation of the P2z-purinoceptor for extracellular ATP in dissociated rat parotid cells is presented in studies using compounds that inhibit protein kinases. Preincubation of acinar cells with the protein kinase catalytic-site inhibitors K-252a and staurosporine, as well as with the regulatory-domain inhibitor sphingosine, specifically potentiates the elevation in cytosolic Ca2+ concentration ([Ca2+]i) mediated by extracellular ATP, but has no effect on the [Ca2+]i elevation mediated by muscarinic receptors through phospholipase C activation. Phorbol dibutyrate (PDBu), which activates protein kinase C (PKC), has no modulatory effect on ATP-mediated [Ca2+]i elevation. Further, pretreatment with PDBu does not reverse or block the effects of K-252a or sphinogosine, arguing against the involvement of PKC. Other pharmacological manipulations indicate that neither calmodulin-dependent nor cyclic-AMP-dependent kinases are involved. Neither the peak intracellular Ca2+ mobilization nor the sustained Ca2+ entry in response to carbachol or to a Ca2+ ionophore (4-bromo-A23187) is altered by the kinase inhibitors that potentiate the [Ca2+]i response to ATP, indicating that effects on the ATP response are not due to non-specific permeability changes, nor to decreased Ca2+ removal from the cytosol. ATP-mediated influx of Mn2+ as well as ATP-induced membrane depolarization are potentiated in cells preincubated with K-252a, directly demonstrating that cation influx is enhanced through a P2z-specific route. These results show that P2z responses (or purinoceptors) can be modulated and suggest that phosphorylation events are involved.

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Sweet Taste Transduction in Hamster: Role of Protein Kinases

Journal of Neurophysiology ◽

10.1152/jn.2000.83.5.2526 ◽

2000 ◽

Vol 83 (5) ◽

pp. 2526-2532 ◽

Cited By ~ 25

Author(s):

Brian Varkevisser ◽

Sue C. Kinnamon

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Sweet Taste ◽

Taste Buds ◽

Regulatory Proteins ◽

Camp Phosphodiesterase ◽

Kinase C ◽

Taste Transduction ◽

Second Messenger Pathways

Two different second-messenger pathways have been implicated in sweet taste transduction: sugars produce cyclic AMP (cAMP), whereas synthetic sweeteners stimulate production of inositol 1,4,5-tris-phosphate (IP3) and diacylglycerol (DAG). Both sugars and sweeteners depolarize taste cells by blocking the same resting K+conductance, but the intermediate steps in the transduction pathways have not been examined. In this study, the loose-patch recording technique was used to examine the role of protein kinases and other downstream regulatory proteins in the two sweet transduction pathways. Bursts of action currents were elicited from ∼35% of fungiform taste buds in response to sucrose (200 mM) or NC-00274–01 (NC-01, 200 μM), a synthetic sweetener. To determine whether protein kinase C (PKC) plays a role in sweet transduction, taste buds were stimulated with the PKC activator PDBu (10 μM). In all sweet-responsive taste buds tested ( n = 11), PDBu elicited burst of action currents. In contrast, PDBu elicited responses in only 4 of 19 sweet-unresponsive taste buds. Inhibition of PKC by bisindolylmaleimide I (0.15 μM) resulted in inhibition of the NC-01 response by ∼75%, whereas the response to sucrose either increased or remained unchanged. These data suggest that activation of PKC is required for the transduction of synthetic sweeteners. To determine whether protein kinase A (PKA) is required for the transduction of sugars, sweet responses were examined in the presence of the membrane-permeant PKA inhibitor H-89 (10 and 19 μM). Surprisingly, H-89 did not decrease responses to either sucrose or NC-01. Instead, responses to both compounds were increased in the presence of the inhibitor. These data suggest that PKA is not required for the transduction of sugars, but may play a modulatory role in both pathways, such as adaptation of the response. We also examined whether Ca2+-calmodulin dependent cAMP phosphodiesterase (CaM-PDE) plays a role in sweet taste transduction, by examining responses to sucrose and synthetic sweeteners in the presence of the CaM-PDE inhibitor W-7 (100 μM). Inhibition resulted in an increase in the response to sucrose, whereas the response to NC-01 remained unchanged. These data suggest that the pathways for sugars and sweeteners are negatively coupled; the Ca2+ that is released from intracellular stores during stimulation with synthetic sweeteners may inhibit the response to sucrose by activation of CaM-PDE.

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Regulation of NaCl entry into Necturus gallbladder epithelium by protein kinase C

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.2.c531 ◽

1994 ◽

Vol 266 (2) ◽

pp. C531-C535 ◽

Cited By ~ 14

Author(s):

R. Dausch ◽

K. R. Spring

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Kinase Inhibitors ◽

Apical Membrane ◽

Cell Swelling ◽

Nacl Transport ◽

Control Value ◽

Necturus Gallbladder ◽

Kinase C

The role of protein kinase C in the regulation of the mode of NaCl entry into Necturus gallbladder epithelial cells was determined from the rate and magnitude of ouabain-induced cell swelling in the presence of inhibitors. Stimulation of protein kinase C by phorbol ester increased the rate of cell swelling from the control value of 2.9% to 4.7%/min and caused the predominant apical membrane transport mechanism for NaCl to switch from bumetanide-sensitive Na-Cl cotransport to amiloride-sensitive parallel exchange. Na-Cl cotransport could be restored as the predominant mode of NaCl entry by treatment of stimulated tissues with the kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and calphostin C. Therefore the mechanism of NaCl transport across the apical membrane can be controlled by the activity of protein kinase C.

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Modification of Ca2+-handling in cardiomyocytes by redox sensitive mechanisms in response to ouabain

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2012-0215 ◽

2013 ◽

Vol 91 (1) ◽

pp. 45-55 ◽

Cited By ~ 1

Author(s):

Harjot K. Saini-Chohan ◽

Larry Hryshko ◽

Yan-Jun Xu ◽

Naranjan S. Dhalla

Keyword(s):

Signal Transduction ◽

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinases ◽

Redox Status ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Kinase C ◽

Transduction Mechanisms

We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca2+]i produced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca2+]i, augmented the KCl-induced increase in [Ca2+]i, and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-l-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca2+], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca2+]i. Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca2+]i, whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na +–K+ ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca2+-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca2+]i in response to ouabain.

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