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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 19, 2003 as doi:10.1096/fj.03-0647fje. |
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Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA
2Correspondence: Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970, USA. E-mail: rdh3{at}cwru.edu
SPECIFIC AIMS
Enhancement of the L-type Ca2+ current (ICa,L) in response to ß-adrenergic receptor (ß-AR) stimulation is one of the main signaling mechanisms contributing to an increase in cardiac output during stress and exercise. However, this mechanism is known to be significantly impaired in heart failure. Failing myocardium is also characterized by an upregulation in the activity of protein kinase C (PKC). The objective of the present study was to investigate the effect of PKC activation on ß1-adrenergic regulation of ICa,L responses in cardiac myocytes.
PRINCIPAL FINDINGS
1. PKC decreases ICa,L sensitivity to stimulation by Gs coupled receptors
To determine what effect, if any, activation of PKC has on ß-adrenergic responses we used adult guinea-pig ventricular myocytes and compared the action of the ß-AR agonist isoproterenol (Iso) on the ICa,L in control myocytes and myocytes treated with the PKC agonist phorbol-12,13-dibutyrate (PDBu) (Fig. 1
A–C). Under control conditions Iso produced a concentration-dependent increase in ICa,L amplitude with an EC50 of 1.0 ± 0.1 nM. However, in myocytes treated with 100 nM PDBu, the EC50 increased to 3.0 ± 0.1 nM. This represents a threefold decrease in the sensitivity to Iso (P<0.001). This effect of PDBu was not mimicked by the inactive analog 4
-phorbol-12,13-didecanoate (4-PDD). In the presence of 100 nM 4-PDD, Iso stimulated the ICa,L with an EC50 of 1.1 ± 0.1 nM, which was not significantly different from that observed in the absence of phorbol ester (P>0.5). These results suggest that activation of PKC is associated with a decrease in the sensitivity of the ICa,L to ß-AR stimulation.
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Unlike its effect on the response to Iso, PDBu treatment did not significantly affect the ICa,L response to forskolin, an agonist which directly stimulates adenylyl cyclase, bypassing the requirement for activation of either ß-ARs or the stimulatory G protein, Gs (Fig. 1D
). In the absence of PDBu, forskolin stimulated the ICa,L with an EC50 of 247 ± 55.6 nM. This is not significantly different (P>0.5) from the EC50 of 206 ± 48.4 nM observed in PDBu-treated myocytes. This result supports the hypothesis that the reduction in ß-adrenergic sensitivity is not due to PKC acting at a point down stream of adenylyl cyclase activation.
On the other hand, activation of PKC did significantly decrease the ICa,L response to histamine. In guinea pig ventricular myocytes, the ICa,L can be stimulated by activation of H2 histamine receptors through Gs dependent regulation of adenylyl cyclase (Fig. 1E
). In the absence of phorbol ester, histamine increased the ICa,L with an EC50 of 53 ± 3.3 nM, yet in PDBu-treated myocytes, histamine stimulated the current with an EC50 of 142 ± 21 nM. This represents a 2.7 fold decrease in the sensitivity to histamine (P<0.01). Furthermore, this effect of PDBu could be significantly attenuated (P<0.01) by bisindolylmaleimide I (BIM), a specific inhibitor of PKC. In PDBu-treated myocytes exposed to 300 nM BIM, histamine stimulated the ICa,L with the EC50 of 84 ± 14 nM (Fig. 1E
). These results suggest that activation of PKC is associated with reduced responsiveness of the ICa,L to Gs coupled receptors.
2. The decrease in ICa,L sensitivity is associated with facilitation of an inhibitory response
One explanation for the decrease in ICa,L sensitivity to ß-adrenergic and histamine receptor stimulation is that PKC directly attenuates the Gs dependent signaling mechanism. An alternative explanation is that PKC facilitates coupling of the receptor to a parallel inhibitory mechanism that can antagonize the stimulatory response. This latter hypothesis was supported by the finding that PDBu treatment revealed the ability of Iso to directly inhibit stimulation of the ICa,L at higher concentrations (Fig. 1B
). PDBu treatment also facilitated Iso-mediated inhibition of ICa,L stimulated by histamine (Fig. 2
). It was found that Iso (3 µM) actually produced a small (9.6 ±2.5 %, n=11) but significant (P<0.01) inhibition of the ICa,L response to a maximally stimulating concentration of histamine in control cells. However, in the presence of PDBu, Iso inhibited the maximal response to histamine by 34 ± 2.8 % (n=10). The inhibitory effect of Iso was not significantly altered in 4-PDD-treated myocytes (10 ±2.1 %, n=4, P>0.5) or in PDBu-treated myocytes exposed to the PKC inhibitor BIM (9.2 ±1.7 %, n=8, P>0.5). PDBu treatment also facilitated the ability of histamine to inhibit the maximal stimulatory response to Iso.
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3. The same receptor subtype mediates both inhibitory and stimulatory responses
In guinea pig ventricular myocytes, the stimulatory effects that Iso and histamine have on the ICa,L are mediated by ß1-adrenergic and H2 receptors, respectively. Although it is possible that activation of PKC actually facilitates responses mediated by other receptor subtypes, we found that CGP 20712A, a selective ß1-AR antagonist, completely blocked the inhibitory effect of Iso in PDBu treated myocytes, but ICI 118,551, a selective ß2-AR antagonist, had no significant effect. Furthermore, cimetidine, a specific H2 receptor antagonist, completely blocked the inhibitory response to histamine in PDBu treated myocytes, but pyrilamine, a specific H1 receptor antagonist, and thioperamide, a specific H3 receptor antagonist, had no significant effect.
4. PKC facilitates receptor coupling to a pertussis toxin sensitive G protein
In the heart it has been demonstrated that ß2-ARs can activate both Gs and Gi dependent signaling pathways. To evaluate the possibility that activation of PKC promotes the coupling of ß1-adrenergic as well as H2 histamine receptors to Gi, we compared the inhibitory effects of Iso and histamine in PTX and non-PTX-treated myocytes. PTX is known to prevent activation of the inhibitory G proteins Gi and Go. We found that PTX significantly reduced the inhibitory effects of both Iso and histamine in PDBu-treated myocytes. These results support the hypothesis that ß1-adrenergic and H2 histamine receptors can activate both Gs and Gi/o dependent signaling pathways in guinea pig ventricular myocytes.
CONCLUSIONS AND SIGNIFICANCE
Using native cardiac myocytes, we found that activation of PKC regulates coupling of the ß1-AR to a pertussis toxin sensitive G protein, Gi/o. In the heart, the ß1-AR normally elicits responses through a signaling pathway that involves activation of the stimulatory G protein, Gs, and subsequent switching on of adenylyl cyclase. We found that activation of PKC caused the ß1-AR to couple to Gi, which can inhibit adenylyl cyclase. The result was that following activation of PKC, ß1-AR stimulation produced both stimulatory and inhibitory responses (Fig. 3
). This dual coupling was apparent as a significant decrease in the sensitivity of stimulatory responses to submaximal ß1-AR activation as well as attenuation or direct inhibition of stimulatory responses by higher levels of ß1-AR activation. We also discovered that this effect of PKC is not unique to regulation of ß1-adrenergic responses. Activation of PKC also facilitated coupling of H2 histamine receptors to Gi mediated inhibitory responses.
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Both ß1- and ß2-ARs are associated with functional responses linked to Gs-dependent activation of adenylyl cyclase and production of cAMP in cardiac myocytes. However, the ß2-AR is also known to couple to Gi. In fact, it has been suggested that coupling of the ß2-AR to Gi localizes cAMP production to subcellular compartments and helps explain differences in the functional response to ß1 and ß2-AR stimulation. The fact that ß1-AR stimulation is apoptotic, but ß2-AR stimulation is not, has been attributed to the ability of the ß2-AR to couple to Gi.
Despite concerted efforts, no one has been able to demonstrate evidence for coupling of the ß1-AR to Gi in cardiac myocytes. Our results are consistent with the conclusion that under control conditions, evidence of ß1-AR interaction with Gi is not obvious. However, under conditions that activate PKC, the ß1-AR does elicit responses that are consistent with the activation of Gi. Such effects may contribute to the decrease in ß-adrenergic responsiveness of the heart under different pathological conditions; it may also point to new directions for investigating mechanisms underlying G protein coupled receptor (GPCR) mediated responses in the heart.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0647fje 
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), cells treated with 100 nM PDBu (
), and cells treated with either 100 nM 4
) or 100 nM PDBu and 300 nM BIM (
