Energetic Effects of Protein Kinase C Inhibition During Calcium Induced Inotropy in the Isolated Perfused Mouse Heart

Adenosine is a physiologically important nucleoside in the cardiovascular system where it can act as a cardioprotectant and modulator of energy usage. Adenosine transporters (ATs) modulate cellular adenosine levels, which, in turn, can affect a number of processes such as receptor activation and glucose uptake, but their role in cardiac physiology is poorly understood. Therefore, we have developed a new cell model by determining various adenosine-related characteristics of HL-1, an immortalized atrial cardiomyocyte murine cell line. Adenosine uptake in HL-1 cells is sodium independent, saturable, and inhibitable by nucleoside transport inhibitors (nitrobenzylthioinosine (NBTI), dipyridamole, dilazep). Reverse transcription polymerase chain reaction analysis confirmed that HL-1 cells possess mouse equilibrative nucleoside transporters 1 and 2 (mENT1, mENT2) and kinetic analyses indicate moderate-affinity (Km = 51.3 ± 12.9 μM), NBTI-sensitive adenosine transport. NBTI binds at a high-affinity single site (Bmax = 520 ± 10 fmol/mg protein, Kd = 0.11 ± 0.04 nM, 1.6 × 105 NBTI-binding sites/cell). HL-1 cells possess adenosine receptor, metabolic enzyme, protein kinase C isoform, and insulin-stimulated glucose transport profiles that match normal mouse heart. Therefore, HL-1 is an excellent model to study ATs within cardiomyocytes and the first model for evaluating in detail the role of the ATs in modulating effects of adenosine.Key words: adenosine, nucleoside transport, HL-1 cells, cardiovascular, glucose transport, protein kinase C.

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Protein kinase C-δ mediates adenosine A3 receptor-induced delayed cardioprotection in mouse

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00095.2003 ◽

2003 ◽

Vol 285 (1) ◽

pp. H434-H441 ◽

Cited By ~ 31

Author(s):

Ting Cun Zhao ◽

Rakesh C. Kukreja

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Ventricular Function ◽

Nuclear Factor Κb ◽

Mouse Heart ◽

Adenosine A3 Receptor ◽

Kinase C ◽

Computer Morphometry ◽

Delayed Cardioprotection

We investigated the role of protein kinase C in adenosine A3 receptor (A3AR)-induced delayed cardioprotection in the mouse heart. Mice were treated with selective A3AR agonist N6-(3-iodobenzyl)adenosine-5′- N-methyluronamide (IB-MECA). Twenty-four hours later, hearts were perfused in the Langendorff mode and subjected to 30 min of global ischemia and 30 min of reperfusion. Infarct size was determined by computer morphometry of tetrazolium-stained sections, and ventricular function was monitored by inserting a fluid-filled balloon into the left ventricle (LV). Chelerythrine chloride (CHE, 5.0 mg/kg) and rottlerin (Rot, 0.3 mg/kg) were given 30 min before IB-MECA to block total and PKC-δ isoforms, respectively. IB-MECA caused postischemic reduction in necrosis and improvement in ventricular function, which was abolished by CHE. Western blot analysis demonstrated translocation of the PKC-δ isoform but not the α, ϵ, ξ, η isoform(s) from cytoplasm to the membrane fraction after 30 min of IB-MECA administration. A3AR antagonist MRS-1191 and CHE blocked the translocation of PKC-δ. Furthermore, IB-MECA-induced increase in nuclear factor-κB binding was diminished by CHE. These results provide direct evidence of an essential role of PKC, and more specifically, PKC-δ in A3AR-induced delayed cardioprotection.

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?1 connexin (connexin43) gap junctions and activities of cAMP-dependent protein kinase and protein kinase C in developing mouse heart

Developmental Dynamics ◽

10.1002/dvdy.1232 ◽

2002 ◽

Vol 223 (1) ◽

pp. 96-107 ◽

Cited By ~ 12

Author(s):

John C. Duncan ◽

William H. Fletcher

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Gap Junctions ◽

Mouse Heart ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Kinase C ◽

Dependent Protein ◽

Connexin43 Gap Junctions

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Protein kinase C isoform expression and activity in the mouse heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.5.h2062 ◽

2001 ◽

Vol 281 (5) ◽

pp. H2062-H2071 ◽

Cited By ~ 13

Author(s):

Kathy L. Schreiber ◽

Louise Paquet ◽

Bruce G. Allen ◽

Hansjörg Rindt

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Developmental Change ◽

Developmental Time ◽

Mouse Heart ◽

Neonatal Cardiomyocytes ◽

Kinase C ◽

Adult Cardiomyocytes ◽

Pkc Isoforms ◽

Fast Performance Liquid Chromatography

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCα and PKCβII, as well as two Ca2+-independent isoforms, PKCδ and PKCε, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCδ and PKCε were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCα in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCδ and PKCε were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.

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