HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 286/6/H2065    most recent 00933.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Gómez, A. M. Articles by Pappano, A. J. Search for Related Content PubMed PubMed Citation Articles by Gómez, A. M. Articles by Pappano, A. J.

Autonomic regulation of calcium and potassium channels is oppositely modulated by microtubules in cardiac myocytes

¹Physiopathologie Cardiovasculaire, Institut National de la Santé et de la Recherche Médicale U-637, EA-3759, Centre Hospitalier Universitaire Arnaud de Villeneuve, F-34295 Montpellier, France; and ²Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030

Submitted 2 October 2003 ; accepted in final form 16 January 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
We recently showed that colchicine treatment of rat ventricular myocytes increases the L-type Ca²⁺ current (I_Ca) and intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients and interferes with adrenergic signaling. These actions were ascribed to adenylyl cyclase (AC) stimulation after G_s activation by ,-tubulin. Colchicine depolymerizes microtubules into ,-tubulin dimers. This study analyzed muscarinic signals in myocytes with intact or depolymerized microtubules. Myocytes were loaded with the Ca²⁺ indicator fluo 3 and were field stimulated at 1 Hz or voltage clamped. In untreated cells, carbachol (CCh; 1 µM) induced ACh-activated K⁺ current [I_K(ACh)], which happens via -subunits from the activation of G_i. Carbachol also reduced [Ca²⁺]_i transients and contractions. Once G_i is activated by muscarinic agonist, the _i-subunit is released from the -subunits, but it is silent, and its inhibition of the AC/cAMP cascade, manifested by I_Ca reduction, is not seen unless AC has been previously activated. In colchicine-treated cells, CCh caused greater reductions of [Ca²⁺]_i transients and contractions than in untreated cells. The _i-subunit became effective in signaling through the AC/cAMP cascade and reduced I_Ca without changing its voltage-dependence. Isoproterenol (Iso) regained its efficacy and reversed I_Ca inhibition by CCh. Stimulation of I_Ca by forskolin persisted in colchicine-treated cells when Iso was ineffective. The effect of CCh on I_K(ACh) was occluded in colchicine-treated cells. Colchicine treatment, per se, may increase I_K(ACh) by -subunits released from G_s to mask this effect of CCh. Microtubules suppress I_Ca regulation by _i; their disruption releases restraints that unmask muscarinic inhibition of I_Ca. Summarily, colchicine treatment reverses regulation of ventricular excitation-contraction coupling by autonomic agents.

cytoskeleton; excitation-contraction coupling; colchicine

This laboratory reported that, when colchicine had disrupted microtubule structure, the L-type Ca²⁺ current (I_Ca) and intracellular Ca²⁺ transients increased (9). The results of colchicine treatment were similar to those of the -adrenoceptor agonist isoproterenol (Iso), whose stimulation of I_Ca and of intracellular Ca²⁺ transients was blunted in colchicine-treated myocytes. The adenylyl cyclase (AC) inhibitor, 2'-deoxyadenosine 3'-monophosphate (2'd3'-AMP), prevented the increase of I_Ca by colchicine. It was concluded that free tubulin (tubulin dimer) transferred exchangeable GTP to the stimulatory guanine nucleotide binding protein, G_s, to activate AC (see also Ref. 25). Washout of colchicine reversed its effects (14). In intact cells, microtubule disruption by colchicine-modulated Ca²⁺ sparks characteristics (14).

In cardiac myocytes, microtubule disruption could potentially result in activation of other G proteins, by making GTP available, such as the inhibitory guanine nucleotide binding protein G_i (22). In particular, the action of muscarinic receptor (mAChR) agonists mediated by the inhibitory guanine nucleotide binding protein G_i may also be modulated by the cytoskeleton (22). In visceral smooth muscle, carbachol inhibits barium current (I_Ba) and colchicine increased the amplitude of I_Ba and selectively blocked its inhibition by carbachol (21).

Here we analyzed whether muscarinic effects are altered in rat ventricular myocytes by microtubule disruption with colchicine. This cell preparation has several advantages. Muscarinic agonist decreases contractions by activating ACh-activated K⁺ current [I_K(ACh)] (17). The -subunits from G_i are able to activate inwardly rectifying K⁺ channels [I_K(ACh)]. The _i-subunit is selective for inhibition of cardiac AC (20), yet muscarinic agonist is not reported to suppress I_Ca. Our approach allowed a systematic examination of how colchicine reciprocally modulated autonomic signaling by -adrenoceptors and mAChRs in a single cardiac cell type. A preliminary account of our findings has been presented in abstract form (10).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell preparation. Ventricular myocytes were isolated from the hearts of anaesthetized (pentobarbital sodium, 100 mg/kg ip) male Wistar rats (300 g) as described elsewhere (16). After isolation, cells were kept at room temperature (24–25°C) in Tyrode solution (see Electrophysiology) containing 1 mM CaCl₂. Cells were incubated with 1 µM colchicine during at least 2 h, as previously explained (14). All experiments were carried out according to the ethical principles laid down by the French (Ministry of Agriculture) and European Union Council Directives for care of laboratory animals.

Electrophysiology. The whole cell patch-clamp technique was used to study carbachol-activated I_K(ACh) and the L-type I_Ca using a patch-clamp amplifier Axopatch 200A (Axon Instruments). Currents were monitored with pClamp7. For I_K(ACh) measurements, cells were superfused with HEPES-buffered Tyrode solution containing (in mM) 140 NaCl, 4 KCl, 1.8 CaCl₂, 1 MgCl₂, 5.5 glucose, and 5 HEPES (pH = 7.4, with NaOH). The pipette filling solution contained (in mM) 125 K-aspartate, 15 KCl, 1 MgCl₂, 4 EGTA, 3 MgATP, 5 Na₂ phosphocreatine, 0.2 Na₂GTP, and 10 HEPES (pH = 7.2, with KOH). Measurements of I_Ca were made by using intracellular and extracellular Cs⁺-rich solutions to block potassium currents. Extracellular solution contained (in mM) 140 NaCl, 0.5 MgCl₂, 5 CsCl, 5.5 glucose, 5 HEPES, 1.8 CaCl₂ (pH set to 7.4 with NaOH). The pipette was filled with a solution containing (in mM) 130 CsCl, 1 MgCl₂, 1 NaH₂PO₄, 3.6 Na₂ phosphocreatine, 5 MgATP, 10 HEPES, and 4 EGTA (pH fixed at 7.2 with CsOH).

Calcium transients and contractions. In another set of experiments, intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients and associated contractions were recorded in intact myocytes. Cells were incubated in fluo 3-AM (1–3 µM) for 20 min and then placed in Tyrode solution for at least 20 min to allow intracellular esterases to deesterify the dye. Myocytes were then placed in an inverted microscope in a chamber that had platinum electrodes parallel to its long axis to field stimulate the cells. Stimuli (1- to 2-ms duration, voltage at 1.2x threshold) were applied at a frequency of 1 Hz. During experiments, myocytes were superfused with a Tyrode solution of the same composition than the one used to record I_K(ACh) (see above).

Images from fluo 3-loaded myocytes were obtained with a laser scanning confocal microscope (Zeiss 510 LSM), attached to an inverted microscope fitted with a water-immersion objective (x63, 1.2 numerical aperture). Fluo 3 fluorescence was excited at a wavelength of 488-nm by an argon ion laser. Fluorescence emission was measured at wavelengths of 505 nm. Images were acquired in line-scan mode. A single myocyte was repetitively scanned along a line parallel to the longitudinal cell axis every 1.5 ms. Because the line of scan was parallel to the longitudinal axis, the length of the cell could be measured to calculate the percentage of cell shortening from the confocal image. Each image was processed and analyzed with the background subtracted using the TDL (Research System) program. Fluorescence transients were obtained by averaging the fluorescence values within the cell. Amplitude was measured as the maximum value of F/F₀, where F is the fluorescence signal and F₀ is the basal fluorescence (measured as the average of the lowest values on the fluorescence signal). The F/F₀ ratio was converted to intracellular Ca²⁺ ([Ca²⁺]_i) as reported earlier (8) with the expression [Ca²⁺]_i = K_d (F/F_o)/{(K_d/[Ca²⁺]_rest) + 1 – (F/F_o)}, where [Ca²⁺]_rest is initial intracellular Ca²⁺ taken as 125 nM and K_d is the dissociation constant (400 nM) for Ca²⁺ and fluo 3.

To verify the microtubule disruption in our conditions, control and colchicine-treated myocytes were fixed and immunolabeled with anti -tubulin as previously detailed (14). Images were recorded in the same microscope by using 2-photon excitation delivered by a Ti-Sa laser (Coherent) at 800 nm. Emission was collected at >505 nm after filtering infrared light. Three-dimensional reconstructions were made with Imaris (Bitplane AG).

All experiments were performed at room temperature.

Data analysis and statistics. Data are reported as means ± SE. Student's t-test for paired and unpaired samples was used as appropriate. P 0.05 was regarded statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Peak [Ca²⁺]_i and cell shortening. Figure 1A shows representative examples of line-scan images recorded in a control rat cardiac myocyte before and during 1 µM carbachol (CCh) application. Data obtained in a myocyte, where microtubules had been disrupted by colchicine treatment, are shown. The intensity of [Ca²⁺]_i fluorescence was higher in the colchicine-treated cell, and fluorescence intensity decreased in CCh. Pooled data are shown in Fig. 1B. In control conditions, field stimulation at 1 Hz evoked [Ca²⁺]_i transients that averaged 458 ± 31 nM (n = 16). At this peak [Ca²⁺]_i (Fig. 1B), cell shortening averaged 14.2 ± 1.0% of resting cell length. When 1 µM CCh was added, peak [Ca²⁺]_i declined by 8% to 421 ± 34 nM (P < 0.03) and cell shortening diminished by 11% to 12.6 ± 0.7% of cell length (P < 0.001). The reduced cell shortening by muscarinic agonist is consistent with the observations of other studies (17) in rat ventricular myocytes.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Carbachol (CCh, 1 µM) is more effective in decreasing Ca2+ transients and contractions in colchicine-treated myocytes. A: line-scan images obtained during field stimulation (1 Hz) in a control (left) and in a colchicine-treated myocyte (right). Top: before CCh. Bottom: CCh effect. The color-coded bar indicates F/F0 (F being the fluorescence value and F0 the average fluorescence values before electrical stimulation). B: graphical summary of data from control cells (squares, n = 16) and from colchicine-treated cells (diamonds, n = 13) before (open symbols) and after CCh (filled symbols). In control cells, CCh reduced cell shortening significantly (P < 0.05). Peak intracellular Ca2+ concentration ([Ca2+]i) was significantly greater in colchicine-treated cells than in control myocytes (*P < 0.02); cell shortening was not (P = 0.25). CCh reduced peak [Ca2+]i (P < 0.001) and cell shortening (P < 0.001) in colchicine-treated cells.

L-type calcium current. Muscarinic agonists per se do not significantly affect I_Ca in mammalian ventricular myocytes until cAMP has been raised by, for example, -adrenoceptor agonist (accentuated antagonism, Ref. 15). We tested the possible effect of CCh in control myocytes at a higher concentration (10 µM) to ascertain whether muscarinic stimulation can induce some inhibition of the I_Ca. Figure 2A shows sample traces of I_Ca of a control myocyte before and during CCh perfusion and in the presence of the -agonist Iso. The changes in I_Ca are plotted as a function of time. CCh slightly decreased I_Ca by 20%, which could be due to the basal AC activity. Further application of Iso almost doubled the initial I_Ca, because it is characteristic of -adrenergic stimulation. However, the larger-than-normal I_Ca recorded in colchicine-treated myocytes was markedly reduced by 10 µM CCh (Fig. 2B). Peak I_Ca decreased to –10 pA/pF after the 3-min CCh addition. Applying Iso (1 µM) in the presence of CCh largely restored I_Ca to its original amplitude of –14 pA/pF. The variations of I_Ca by CCh and Iso applications in this colchicine-treated cell are shown in Fig. 2B. CCh was more effective in decreasing I_Ca in the colchicine-treated myocyte, and application of Iso restored the reduced I_Ca up to, but not over, the original value before CCh. The average of such experiments in colchicine-treated cells is shown in Fig. 3. Application of Iso alone had no effect on peak I_Ca density, a finding reported previously (9). CCh, per se, inhibited I_Ca by up to 50% in these cells, an effect reversed by the subsequent addition of Iso (Fig. 3). Thus the actions of these agonists appeared to be reversed in colchicine.

View larger version (15K): [in this window] [in a new window]   Fig. 2. CCh inhibits L-type Ca2+ current (ICa) in a colchicine-treated myocyte. A: control. B: colchicine-treated myocyte. Top: voltage-clamp protocol to elicit ICa at 0 mV in cells dialyzed with Cs+-rich pipette solution. Middle: original data recordings before, after 10 µM CCh and after adding 1 µM isoproterenol (Iso) on top of CCh. Bottom: time course of percent change of current.

View larger version (21K): [in this window] [in a new window]   Fig. 3. CCh decreases ICa amplitude in colchicine-treated cells (Colchi) and restores their sensitivity to Iso. A: current-voltage (I-V) relationships of mean normalized ICa density recorded in colchicine-treated myocytes (, solid black line, n = 12). Addition of 10 µM Iso alone (, dashed line, n = 3) had no significant effect on ICa. CCh alone (, dotted line, n = 7) at 10 µM reduced ICa by 50% in colchicine-treated cells. Application of Iso on top of CCh (, dot-dash line, n = 5) restored ICa to initial values seen in colchicine alone. All data are normalized to the respective controls. *P < 0.05, **P < 0.001 compared with control values, P <0.05 compared with CCh values. B: averaged maximal conductance (Gmax) calculated by fitting the I-V curve to the equation y = (x – Vrev)·Gmax·(–1/{1 + exp[(x – V0.5)/Dx]} + 1), where Vrev is the reversal potential, V0.5 is the potential of demiactivation, and Dx is the slope factor of the activation curve (2). *P < 0.05, **P < 0.005.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Forskolin enhances ICa in both control and colchicine-treated ventricular myocytes. A and B: current-voltage relationships of mean ICa density established in control (, n = 5) and colchicine-treated (, n = 6) myocytes. Forskolin (10 µM) increases ICa amplitude by 2.3-fold both in control (, n = 5) and in colchicine-treated (, n = 6) myocytes. Insets: examples of ICa current recordings elicited at 0 mV before (solid line) and during (dashed line) forskolin application. *P < 0.05, **P < 0.001 related to forskolin action. P < 0.05, P < 0.005 between control and colchicine-treated cells.

I_K(ACh). In these experiments, 3-s voltage ramps from –100 to +20 mV were applied; the current-voltage relation displayed inward rectification (Fig. 5, A and B). CCh (1 µM) was added, and the voltage ramp was applied within 5–10 s after a change in holding current was detected. In this cell, the membrane hyperpolarized by 2 mV and membrane current shifted inward at –100 mV. In the absence of colchicine membrane current was –11.8 ± 1.2 pA/pF at –100 mV when normalized to cell capacitance (Fig. 5A). CCh shifted current inward by –0.6 ± 0.1 pA/pF (n = 10) at –100 mV (Fig. 5C). These results indicate the expected activation of inwardly rectifying I_K(ACh) by CCh (17). As the exposure to CCh continued, I_K(ACh) declined because of desensitization.

View larger version (13K): [in this window] [in a new window]   Fig. 5. CCH is unable to activate an inward current in colchicine-treated myocytes. A: currents elicited from –100 to –60 mV in a control myocyte before (black trace) and during (gray trace) 1 µM CCh application. Membrane current became more inward at –100 mV. The result indicates the expected activation of inwardly rectifying IK(ACh) by CCh. The zero current potential was –72.77 mV before CCH and –73.08 during CCH application. B: the same in a colchicine-treated cell. No inward current was activated by CCh. Membrane current changes induced by CCh at –100 (white bars) and +20 mV (gray bars) in control (C; n = 10) and in colchicine (D; n = 5). *P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Microtubule disruption by colchicine augments muscarinic inhibition of excitation-contraction coupling in rat ventricular myocytes. CCh reduced the magnitude of intracellular Ca²⁺ transients and cell shortening more when microtubules had been disrupted. Colchicine treatment unmasks muscarinic inhibition of I_Ca, and this can largely account for the greater suppression of Ca²⁺ transients and contractions. On the contrary, colchicine treatment occludes I_K(ACh) activation by CCh. Also, microtubule disruption interferes with -adrenoceptor regulation of excitation-contraction coupling (9, 14). Thus microtubule depolymerization unmasks muscarinic signaling to L-type Ca²⁺ channels and masks muscarinic signaling to I_K(ACh) in rat ventricular myocytes. The proposed mechanisms of action are described in Fig. 6 in the untreated state, with the microtubule cytoskeleton intact (2-photon 3-D image reconstruction) and in the presence of colchicine, with all microtubules depolymerized. Agonist occupancy of muscarinic and ₁-adrenoceptors would promote dissociation of heterotrimeric G proteins via guanine nucleotide exchange (GTP replaces GDP). In untreated cells, Iso occupies ₁-adrenoceptors and stimulates I_Ca through the _s/AC/cAMP cascade, and CCh occupies mAChR and activates I_K(ACh) through -subunits. CCh does not inhibit I_Ca until Iso has acted. In colchicine-treated cells, ,-tubulin dimers would donate GTP to _s-subunit to activate the AC/cAMP cascade and increase I_Ca. Now, _i-subunit is no longer "silent" and inhibits the AC pathway to I_Ca. -subunits from G_s would have already activated (and desensitized) I_K(ACh) to occlude its activation by CCh. Not shown in the figure is the action of forskolin, which bypasses G_s protein to stimulate AC directly and increase I_Ca.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Top: Three-dimensional reconstructions of 2-photon images of rat cardiomyocytes fixed and immunolabeled with anti--tubulin in control conditions (A) and after 2-h incubation with 1 µM colchicine (B). Cell thickness was 5.2 µm for the control myocyte and 4.4 µm for the colchicine-treated myocyte. The microtubule cytoskeleton is absent in the colchicine-treated myocyte. Bottom: proposed regulation of dihydropyridine receptor (DHPR)-Ca2+ channel and K(ACh) channel by agonists at -adrenoceptors () and muscarinic receptors (M) in absence (A) and presence (B) of colchicine. In control conditions, muscarinic agonist activates K(ACh) via -subunits from Gi; the I-subunit inhibits Ca2+ entry at DHP-sensitive channels only after they have been activated by the -adrenoceptor agonist that stimulates the adenylyl cyclase (AC)/cAMP/PKA cascade (not shown) via the s-subunit of Gs. In colchicine, ,-tubulin donates GTP to AC causing eventual increase in ICa and unmasks suppression of cAMP cascade by muscarinic agonist inhibition of AC. -Subunits from GS may activate K(ACh) channels and interfere with subsequent action of muscarinic agonist. See DISCUSSION for details.

Forskolin, which directly activates AC, retains its ability to increase I_Ca by the same proportion in the absence and presence of colchicine. This indicates that AC sensitivity to stimulation is unchanged and points to a target proximal to AC as the site for diminished Iso effect. By donating GTP to G_s, ,-tubulin dimers activate AC via G_s. Type V AC, which is found in the heart, is activated synergistically by forskolin and _s-subunits but additively by Iso and _s-subunits (reviewed in Ref. 20). Therefore, the ability of _s-GTP arising from agonist occupancy of -adrenoceptors to stimulate AC activity should be diminished in colchicine-treated myocytes (16). The net effect is to occlude stimulation of I_Ca by Iso (Refs. 9, 14, and present report). Dissociated G_s would also reduce -adrenoceptor affinity for agonist (7). In adrenal cortical cells treated with colchicine, forskolin continued to stimulate AC, whereas ACTH, which acts through _s-subunit, no longer did so (6).

Muscarinic agonists minimally inhibit I_Ca in rat ventricular myocytes unless AC has been previously activated (17). Cardiac isoforms of AC, types V and VI, are inhibited by _i- but not by -subunits (20). There is no evidence for negative regulation of I_Ca by -subunits in rat ventricular myocytes in contrast to results when L-type Ca²⁺ channels are expressed in Xenopus oocytes (12). The lack of _i-GTP signaling could arise from inaccessibility of _i-GTP or insufficient AC activity. Colchicine, either by preventing sequestration of _i-subunits or by activating AC through _s-GTP, could remove restraints on this component of muscarinic signaling. We favor the view that by releasing ,-tubulin dimers, colchicine stimulates AC and therefore reveals muscarinic inhibition of I_Ca. Muscarinic inhibition of I_Ca in colchicine-treated myocytes conforms to "accentuated antagonism," because inhibition requires the prior stimulation of AC (15). When _i-GTP is present, AC is inhibited and thus the cAMP/PKA phosphorylation cascade. Conceivably, ,-tubulin dimers could also donate GTP to the -subunit of the inhibitory guanine nucleotide binding protein, G_i (18). In rat ventricular myocytes, the evidence indicates that microtubule disruption leads to activation of G_s but not of G_i. This specificity could be explained by the presence of a spatial restriction or compartment between microtubules and G_s.

Colchicine inhibits binding to and activation of mAChR by antilaminin I_gG (1). Our results indicate that colchicine did not impede activation of mAChR by CCh, because I_Ca inhibition by CCh was unmasked in colchicine-treated myocytes and suppression of [Ca²⁺]_i transients and contractions was greater. CCh, like ACh (17), reduces cell shortening in untreated rat ventricular myocytes. This is ascribed to a briefer action potential duration by I_K(ACh) activation. These ligand-gated K⁺ channels are present in ventricle at lower density than in atrium. Rat ventricular myocytes have m₂AChR and K_(ACh) channels with the former in greater abundance (5). The m₂AChR is located primarily on the cell surface and much less so in T-tubules. In contrast, the proteins that comprise K_(ACh) channels (Kir3.1 and Kir3.4) are found in T-tubules. Activation of I_K(ACh) in ventricular myocytes, as in atrial myocytes, presumably arises from the action of -subunits (24). Therefore, muscarinic signaling in myocytes with intact microtubules already involves one component of heterotrimeric G_i. The failure to inhibit I_Ca under these conditions cannot be readily explained by a lack of _i-GTP but rather by its inefficient signaling through AC.

The addition of CCh produces two signaling moieties, because heterotrimeric G_i dissociates into - and -subunits in the presence of GTP. Why is the effect of CCh on I_K(ACh) not increased when colchicine disrupts microtubules? Colchicine, by releasing ,-tubulin dimers, may have already activated I_K(ACh) through -subunits that dissociate from G_s. Thus I_K(ACh) activation by CCh could be occluded. -subunits lack specificity and there is little distinction between them for this signal pathway (23).

How selective is colchicine action? Class C L-type Ca²⁺ channels have an A kinase anchoring protein, microtubule-associated protein 2B (MAP2B), that binds to the ₁-subunit (4). However, these L-type Ca²⁺ channels do not bind tubulin, and microtubule disruption had no effect on MAP2B association with the channel. Cytochalasins B and D prevent actin polymerization, displace G_i, and impede muscarinic signaling to L-type Ca²⁺ channels and K_ACh channels in embryonic stem cells (3). Cytochalasin D, unlike colchicine, reduced I_Ca but did not prevent Iso from stimulating this current (19). Therefore, microtubule disruption by colchicine does not need to involve a direct action on the Ca channel, a redistribution of G_i, or depolymerization of the actin component of the cytoskeleton. Paclitaxel, a microtubule stabilizer, had no effect on I_Ca in rat ventricular myocytes (9) or on cAMP and I_Ca in guinea pig ventricular myocytes (11, 16). Whereas the effects of Iso on cAMP and I_Ca were unchanged in paclitaxel, it opposed muscarinic agonist action on these variables (16).

In summary, microtubule disruption with colchicine reverses the pattern of autonomic signaling in cardiac myocytes. When soluble tubulin is increased, muscarinic agonist is enabled to inhibit I_Ca, whereas -adrenoceptor agonist is disabled from doing so. The reversal of autonomic agonist action can be accounted for by the donation of GTP from soluble tubulin to the -subunit of G_s as well as the -subunit to I_K(ACh). The cytoskeleton is viewed as integrating convergent signal pathways in space and time (13). This concept is well illustrated by the dynamic interplay of tubulin and tubulin dimers on the reactivity of the heart to muscarinic and adrenergic agonists.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by Fondation de France, Association pour la Recherche contre le Cancer, and by National Heart, Lung, and Blood Institute Grant HL-13339 (to A. J. Pappano). B.-G. Kerfant was a fellow from Fondation pour la Recherche Médicale Française. A. M. Gómez is a Centre National de la Recherche Scientifique scientist.

	ACKNOWLEDGMENTS

 
Present address of B.-G. Kerfant: Depts. of Physiology and Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. M. Gómez, Physiopathologie Cardiovasculaire, Institut National de la Santé et de la Recherche Médicale U-637, Centre Hospitalier Universitaire Arnaud de Villeneuve, F-34295 Montpellier, France (E-mail: agomez{at}montp.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Bacman S, Borda E, Denduchis B, Lustig L, and Sterin-Borda L. Participation of cytoskeleton in the effect of antilaminin IgG on cardiac cholinoceptors. Br J Pharmacol 111: 271–277, 1994.[ISI][Medline]
2. Bénitah JP and Vassort G. Aldosterone upregulates Ca²⁺ current in adult rat cardiomyocytes. Circ Res 85: 1139–1145, 1999.[Abstract/Free Full Text]
3. Bloch W, Fan Y, Han J, Xue S, Schöneberg T, Ji G, Lu ZJ, Walther M, Fässler R, Hescheler J, Addicks K, and Fleischmann BK. Disruption of cytoskeletal integrity impairs G_i-mediated signaling due to displacement of G_i proteins. J Cell Biol 154: 753–762, 2001.[Abstract/Free Full Text]
4. Davare MA, Dong F, Rubin CS, and Hell JW. The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons. J Biol Chem 274: 30280–30287, 1999.[Abstract/Free Full Text]
5. Dobrzynski H, Marples DDR, Musa H, Yamanushi TT, Henderson Z, Takagishi Y, Honjo H, Kodama I, and Boyett MR. Distribution of the muscarinic K⁺ channel proteins, Kir3.1 and Kir3.4, in the ventricle, atrium and sinoatrial node of heart. J Histochem Cytochem 49: 1221–1234, 2001.[Abstract/Free Full Text]
6. Feuilloley M and Vaudry H. Role of the cytoskeleton in adrenocortical cells. Endocr Rev 17: 269–288, 1996.[CrossRef][ISI][Medline]
7. Gether U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr Rev 21: 90–113, 2000.[Abstract/Free Full Text]
8. Gómez AM, Cheng H, Lederer WJ, and Bers DM. Ca²⁺ diffusion and sarcoplasmic reticulum transport both contribute to [Ca²⁺]_i decline during Ca²⁺ sparks in rat ventricular myocytes. J Physiol 496: 575–581, 1996.[ISI][Medline]
9. Gómez AM, Kerfant BG, and Vassort G. Microtubule disruption modulates Ca²⁺ signaling in rat cardiac myocytes. Circ Res 86: 30–36, 2000.[Abstract/Free Full Text]
10. Gómez AM, Kerfant BG, Vassort G, and Pappano AJ. Microtubules modulate calcium signals and their regulation by autonomic agonists in cardiac myocytes (Abstract). Biophys J 84: 96a, 2003.
11. Howarth FC, Calaghan SC, Boyett MR, and White E. Effect of the microtubule polymerizing agent taxol on contraction, Ca²⁺ transient and L-type Ca²⁺ current in rat ventricular myocytes. J Physiol 516: 409–419, 1999.[Abstract/Free Full Text]
12. Ivanina T, Blumenstein Y, Shistik E, Barzilai R, and Dascal N. Modulation of L-type Ca²⁺ channels by G and calmodulin via interactions with N and C termini of _1C. J Biol Chem 275: 39846–39854, 2000.[Abstract/Free Full Text]
13. Janmey PA. The cytoskeleton, and cell signaling: component localization and mechanical coupling. Physiol Rev 78: 763–781, 1998.[Abstract/Free Full Text]
14. Kerfant BG, Vassort G, and Gómez AM. Microtubule disruption by colchicine reversibly enhances calcium signaling in intact rat cardiac myocytes. Circ Res 88: e59–e65, 2001.[Abstract/Free Full Text]
15. Levy M. Sympathetic-parasympathetic interactions in the heart. Circ Res 29: 437–445, 1971.[Free Full Text]
16. Malan D, Gallo MP, Bedendi I, Biasin C, Levi RC, and Alloatti G. Microtubules mobility affects the modulation of L-type I_Ca by muscarinic and -adrenergic agonists in guinea pig cardiac myocytes. J Mol Cell Cardiol 35: 195–206, 2003.[CrossRef][ISI][Medline]
17. McMorn SO, Harrison SM, Zang WJ, Yu XJ, and Boyett MR. A direct negative inotropic effect of acetylcholine on rat ventricular myocytes. Am J Physiol Heart Circ Physiol 265: H1393–H1400, 1993.[Abstract/Free Full Text]
18. Roychowdhury S, Wang N, and Rasenick MM. G protein binding and G protein activation by nucleotide transfer involve distinct domains on tubulin: regulation of signal transduction by cytoskeletal elements. Biochemistry 32: 4955–4961, 1993.[CrossRef][Medline]
19. Rueckschloss U, and Isenberg DG. Cytochalsin reduces Ca²⁺ currents via cofilin-activated depolymerization of F-actin in guinea pig cardiomyocytes. J Physiol 537: 363–370, 2001.[Abstract/Free Full Text]
20. Sunahara RK, Dessauer C, and Gilman AG. Complexity and diversity of mammalian adenylyl cyclases. Annu Rev Pharmacol Toxicol 36: 461–480, 1996.[CrossRef][ISI][Medline]
21. Unno T, Komori S, and Ohashi H. Microtubule cytoskeleton involvement of muscarinic suppression of voltage-gated calcium channel current in guinea pig ileal smooth muscle. Br J Pharmacol 127: 1703–1711, 1999.[CrossRef][ISI][Medline]
22. Wang N, Yan K, and Rasenick MM. Tubulin binds specifically to the signal-transducing proteins, G_s and G_i1. J Biol Chem 265: 1239–1242, 1990.[Abstract/Free Full Text]
23. Wickman K, Krapivinsky G, Corey S, Kennedy M, Nemec J, Medina I, and Clapham DE. Structure, G protein activation, and functional relevance of the cardiac G protein-gated K⁺ channel, I_KACh. Ann NY Acad Sci 868: 386–398, 1999.[Abstract/Free Full Text]
24. Yamada M, Inanobe A, and Kurachi Y. G protein regulation of potassium ion channels. Pharmacol Rev 50: 723–760, 1998.[Abstract/Free Full Text]
25. Yan K, Popova JS, Moss A, Shah B, and Rasenick MM. Tubulin stimulates AC activity in C6 glioma cells by bypassing the -adrenergic receptor: a potential mechanism of G protein activation. J Neurochem 76: 182–190, 2001.[CrossRef][ISI][Medline]

This article has been cited by other articles:

				J.-B. Shen and A. J. Pappano An Estrogen Metabolite, 2-Methoxyestradiol, Disrupts Cardiac Microtubules and Unmasks Muscarinic Inhibition of Calcium Current J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 507 - 512. [Abstract] [Full Text] [PDF]

				A. Ouvrard-Pascaud, Y. Sainte-Marie, J.-P. Benitah, R. Perrier, C. Soukaseum, A. N. D. Cat, A. Royer, K. Le Quang, F. Charpentier, S. Demolombe, et al. Conditional Mineralocorticoid Receptor Expression in the Heart Leads to Life-Threatening Arrhythmias Circulation, June 14, 2005; 111(23): 3025 - 3033. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 286/6/H2065    most recent 00933.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Gómez, A. M. Articles by Pappano, A. J. Search for Related Content PubMed PubMed Citation Articles by Gómez, A. M. Articles by Pappano, A. J.