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. 2009 Oct 15;587(Pt 20):4785-97.
doi: 10.1113/jphysiol.2009.179226. Epub 2009 Aug 24.

Laminin enhances beta(2)-adrenergic receptor stimulation of L-type Ca(2+) current via cytosolic phospholipase A(2) signalling in cat atrial myocytes

M R Pabbidi  1 , X Ji, A M Samarel, S L Lipsius
Affiliations

Laminin enhances beta(2)-adrenergic receptor stimulation of L-type Ca(2+) current via cytosolic phospholipase A(2) signalling in cat atrial myocytes

M R Pabbidi et al. J Physiol. .

Abstract

We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN), decreases adenylate cyclase (AC)/cAMP and increases beta(2)-adrenergic receptor (AR) stimulation of L-type Ca(2+) current (I(Ca,L)). This study therefore sought to determine whether LMN enhances beta(2)-AR signalling via a cAMP-independent mechanism, i.e. cytosolic phospholipase A(2) (cPLA(2)) signalling. Studies were performed on acutely isolated atrial myocytes plated on uncoated coverslips (LMN) or coverslips coated with LMN (+LMN). As previously reported, 0.1 microm zinterol (zint-beta(2)-AR) stimulation of I(Ca,L) was larger in +LMN than LMN myocytes. In +LMN myocytes, zint-beta(2)-AR stimulation of I(Ca,L) was inhibited by inhibition of cPLA(2) by arachidonyltrifluoromethyl ketone (AACOCF(3); 10 microm), inhibition of G(i) by pertussis toxin and chelation of intracellular Ca(2+) by 10 microm BAPTA-AM. In contrast to zinterol, stimulation of I(Ca,L) by fenoterol (fen-beta(2)-AR), a beta(2)-AR agonist that acts exclusively via G(s) signalling, was smaller in +LMN than LMN myocytes. Arachidonic acid (AA; 5 microm) stimulated I(Ca,L) to a similar extent in LMN and +LMN myocytes. Inhibition of cAMP-dependent protein kinase A (cAMP/PKA) by either 5 mum H89 or 1 microm KT5720 in LMN myocytes mimicked the effects of +LMN myocytes to enhance zint-beta(2)-AR stimulation of I(Ca,L), which was blocked by 10 microm AACOCF(3). In contrast, H89 inhibited fen-beta(2)-AR stimulation of I(Ca,L), which was unchanged by AACOCF(3). Inhibition of ERK1/2 by 1 microm U0126 inhibited zint-beta(2)-AR stimulation of I(Ca,L) in +LMN myocytes and LMN myocytes in which cAMP/PKA was inhibited by KT5720. In LMN myocytes, cytochalasin D prevented inhibition of cAMP/PKA from enhancing zint-beta(2)-AR stimulation of I(Ca,L). We conclude that LMN enhances zint-beta(2)-AR stimulation of I(Ca,L) via G(i)/ERK1/2/cPLA(2)/AA signalling which is activated by concomitant inhibition of cAMP/PKA signalling and dependent on the actin cytoskeleton. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can remodel beta(2)-AR signalling in atrial muscle.

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Figures

Figure 1
Figure 1. Effects of 10 μm AACOCF3 on zint-β2-AR stimulation of ICa,L in −LMN and +LMN atrial myocytes
A and B, original recordings of ICa,L in −LMN (A) and +LMN (B) atrial myocytes. In −LMN myocytes, zint-induced stimulation of ICa,L was similar in the absence (Aa) and presence (Ab) of AACOCF3. Control zint-β2-AR stimulation of ICa,L was greater on +LMN (Ba) than −LMN (Aa) myocytes. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was inhibited by AACOCF3 (Bb) compared to control (Ba). C and D, bar graphs summarizing the data show that AACOCF3 had no effect on zint-β2-AR stimulation of ICa,L in −LMN myocytes (C) and AACOCF3 significantly decreased zint-β2-AR stimulation of ICa,L in +LMN myocytes (D). Bar graphs in each figure (Figs 1–7) show peak current densities of basal ICa,L (open bars) and agonist-stimulated ICa,L (shaded bars). Numbers in parentheses indicate the number of cells studied. *P < 0.05.
Figure 7
Figure 7. Effects of 10 μm cytochalasin D on zint-β2-AR stimulation of ICa,L in −LMN atrial myocytes
A, zint-β2-AR stimulation of ICa,L was significantly greater in cells treated with cytochalasin D compared with control cells. B, H-89 (5 μm) enhanced zint-β2-AR stimulation of ICa,L compared to control responses in the absence of H-89 (A). In −LMN myocytes treated with cytochalasin D, H-89 failed to enhance zint-β2-AR stimulation of ICa,L. *P < 0.05.
Figure 2
Figure 2. Zint-β2-AR stimulation of ICa,L via cPLA2 signaling is mediated via PTX-sensitive Gi-signaling and reqiures intracellular Ca2+ release
A, effects of pertussis toxin (PTX) on zint-β2-AR stimulation of ICa,L in −LMN and +LMN atrial myocytes. In −LMN myocytes, PTX significantly increased zint-β2-AR stimulation of ICa,L. In +LMN myocytes, PTX significantly decreased zint-β2-AR stimulation of ICa,L. B, effects of 0.1 μm fenoterol (Fen) β2-AR stimulation of ICa,L in −LMN and +LMN atrial myocytes. Fen-β2-AR stimulation of ICa,L was significantly smaller in +LMN than −LMN myocytes. C, effects of 10 μm BAPTA-AM on zint-β2-AR stimulation of ICa,L in −LMN and +LMN atrial myocytes. In −LMN myocytes, zint-β2-AR stimulation was unchanged by BAPTA. In +LMN myocytes, BAPTA significantly decreased zint-β2-AR stimulation of ICa,L compared to control responses. *P < 0.05.
Figure 3
Figure 3. Effects of 5 μm H-89 on zint-β2-AR stimulation of ICa,L in the absence and presence of 10 μm AACOCF3
A–C, original recordings of ICa,L from −LMN atrial myocytes. D, bar graphs summarize data obtained in A–C. Compared to control (A), 5 μm H-89 decreased basal ICa,L and enhanced zint-β2-AR stimulation of ICa,L (B). Zint-β2-AR stimulation of ICa,L was inhibited by the addition of 10 μm AACOCF3 (+ H-89) (C) compared to either control (A) or H-89 alone (B). D, summary graph indicates that zint-β2-AR stimulation of ICa,L was significantly increased by H-89 and significantly inhibited by AACOCF3 (+ H-89) compared to H-89 alone. *P < 0.05.
Figure 4
Figure 4. Effects of 1 μm KT5720 (KT) on zint-β2-AR stimulation of ICa,L in the absence and presence of 10 μm AACOCF3
A–C, original recordings of ICa,L from −LMN atrial myocytes. D, bar graphs summarize data obtained in A–C. Compared to control (A), KT decreased basal ICa,L and enhanced zint-β2-AR stimulation of ICa,L (B). Zint-β2-AR stimulation of ICa,L was inhibited by the addition of 10 μm AACOCF3 (+ KT) (C) compared to either control (A) or H-89 alone (B). D, summary graph indicates that zint-β2-AR stimulation of ICa,L was significantly increased by KT and significantly inhibited by AACOCF3 (+ KT) compared to KT alone. *P < 0.05.
Figure 5
Figure 5. Effects of 5 μm H-89 on fenoterol (Fen)-β2-AR stimulation of ICa,L in the absence and presence of 10 μm AACOCF3
A–C, original recordings of ICa,L from −LMN atrial myocytes. D, bar graphs summarize data obtained in A–C. In control, fen-β2-AR stimulation increased ICa,L. H-89 decreased basal ICa,L and significantly inhibited fen-β2-AR stimulation of ICa,L. Addition of AACOCF3 (+ H-89) had no additional inhibitory effect on fen-β2-AR stimulation of ICa,L compared to H-89 alone. *P < 0.05.
Figure 6
Figure 6. Effects of 1 μm U0126 on zint-β2-AR stimulation of ICa,L
A, in −LMN myocytes, U0126 had no significant effects on zint-β2-AR stimulation of ICa,L. B, in −LMN myocytes, U0126 significantly inhibited the effect of 1 μm KT5720 (KT) to enhance zint-β2-AR stimulation of ICa,L. C, in +LMN myocytes, U0126 also significantly inhibited zint-β2-AR stimulation of ICa,L. *P < 0.05.
Figure 8
Figure 8. Schematic diagram showing the proposed signalling mechanisms responsible for the effects of laminin to enhance β2-AR stimulation of ICa,L in atrial myocytes
Zinterol stimulates β2-ARs coupled to both Gs and Gi signalling pathways. A, in −LMN myocytes β2-ARs act via Gs/AC/cAMP/PKA to stimulate ICa,L. Stimulation of cAMP/PKA also inhibits ERK1/2 signalling preventing activation of cPLA2. B, in +LMN myocytes laminin binding to β1-integrins activates focal adhesion kinase (FAK)/phosphatidylinositol-3′ kinase (PI-(3)K)/protein kinase B (Akt) signalling which inhibits AC/cAMP/PKA activity. Inhibition of cAMP/PKA stimulates ERK1/2 which enhances β2-AR stimulation of ICa,L via activation of Gi-coupled cPLA2/AA signalling.
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