1Enteric NeuroScience Program, Mayo Clinic and Mayo Clinic College of Medicine, Rochester; 2Department of Physiology, College of Medicine, Chung-Ang University, Seoul, Korea; and 3Gastrointestinal Unit, Mayo Clinic and Mayo Clinic College of Medicine, Rochester, Minnesota
Submitted 5 May 2004 ; accepted in final form 11 August 2004
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ABSTRACT |
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ion channels; gases; human studies; patch clamp
Carbon monoxide (CO) is a low molecular weight gas that shares similar properties with another low molecular weight gas, nitric oxide (NO). CO, like NO, is generated under physiological conditions (26). The synthetic enzymes that produce CO, heme oxygenase 1 (HO1) and heme oxygenase 2 (HO2), are widely expressed in the gastrointestinal tract; thus CO is endogenously produced in the gastrointestinal tract (15, 27, 28). In the gastrointestinal tract, CO mediates nonadrenergic noncholinergic neurotransmission (43), sets the smooth muscle membrane potential gradient (43), and appears to protect against the development of postoperative ileus (29).
Both CO and NO activate guanylyl cyclase resulting in the generation of cGMP (34). However, the affinity of CO to guanylyl cyclase is severalfold lower than for NO, suggesting that the effects of CO on guanylyl cylase may require a sensitizing molecule (37) or that other pathways may be involved. Both CO and NO can directly modulate ion channels. NO directly modulates the -subunit, and CO modulates the
-subunit of large conductance Ca2+-activated K+ channels (42). NO also modulates L-type Ca2+ channel activity (4, 38, 44). The effects of NO on L-type Ca2+ channels are complex and tissue dependent. In the gastrointestinal tract, NO inhibits L-type Ca2+ channels from guinea pig Taenia coli (23) and canine gastric circular smooth muscle (31) but activates longitudinal smooth muscle L-type Ca2+ channels in rat intestine (39) and rat fundic longitudinal muscle accompanied by relaxation (14), likely reflecting the dual roles of Ca2+ as a second messenger and as an initiator of contraction. Recently, CO was shown to decrease prenatal rat ventricular myocyte transient outward current density and activate L-type Ca2+ channels (36) postnatally, suggesting that CO, like NO, may regulate L-type Ca2+ channel expression and/or function. The aim of the present study was to determine whether CO modulates intestinal smooth muscle L-type Ca2+ channels and whether the mechanism of action involves the NO synthetic pathway. We found that low levels of exogenous CO activated intestinal L-type Ca2+ channels through activation of NO synthase (NOS), and increased levels of NO and cGMP but not through PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of phosphodiesterase (PDE) III.
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MATERIALS AND METHODS |
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Human jejunal tissue, use of which was approved by the Institutional Review Board, was obtained as surgical waste tissue during gastric bypass operations performed for morbid obesity in otherwise healthy subjects. Tissue specimens were harvested directly into chilled buffer solution with warm ischemia times of 30 s. Single, isolated circular smooth muscle cells were obtained from the human jejunal specimens as described previously (7). The freshly isolated cells were used for electrophysiological recording within 6 h of dissociation.
Transfection of HEK-293 Cells with L-type Calcium Channel Subunits
The human jejunal 1C(CaV1.2)- and
2-subunits were subcloned as an AgeI/NotI fragment from the cDNA library pSPORT1 (Invitrogen, Carlsbad, CA) cloning vector into pEGFP-C1 (Clontech, Palo Alto, CA) as previously published (24). Lipofectamine 2000 Reagent (Invitrogen) was used to transiently express the Ca2+ channel subunits in HEK-293 cells (American Type Culture Collection, Manassas, VA). Transfected cells were identified by their fluorescence microscopy and were patch clamped 4856 h after transfection.
Patch-Clamp Recordings
Whole cell patch-clamp recordings were obtained by using Kimble KG-12 glass pulled on a P-87 puller (Sutter Instruments, Novato, CA). Electrodes were coated with R6101 (Dow Corning, Midland, MI) and fire polished to a final resistance of 3 to 5 M. Currents were amplified, digitized, and processed by using a Axopatch 200A amplifier, a Digidata 1200, and pCLAMP 9 software (Axon Instruments, Foster City, CA). Data were filtered at 5 kHz with an eight-pole Bessel filter. The junction potential between pipette solution and bath solution was electronically adjusted to zero. Access resistance was recorded, and data was only used if access did not change by >5 M
throughout the experiment. Records were obtained in both standard whole cell mode and amphotericin perforated, patch-clamp mode (see RESULTS). For standard whole cell records, rundown of the L-type Ca2+ current was evident within 1015 min of recording. Rundown in amphotericin perforated, patch-clamp experiments was less marked. The time from application of CO to peak effect was typically <90 s. Whereas rundown may have masked the full effects of CO on the L-type Ca2+ current, the lack of a difference in the percent increase in current induced by CO between standard whole cell and amphotericin perforated patch-clamp experiments (see RESULTS) suggests that the effect of rundown on the observed results was not significant. Cells were held at 100 mV and pulsed for 512 ms in 13 steps to voltages ranging from 90 to +30 mV. Cells were returned to 100 mV between pulses with an interpulse interval of 1 s to allow complete recovery from inactivation. Current-voltage (IV) curves were constructed by using the maximal peak inward current amplitude at each voltage. Data were normalized by using the formula Inormv = 100(Iv)/(Imax), where Inormv is the normalized peak inward current at a particular voltage sweep, Iv is the current at each voltage sweep, and Imax is the maximum peak inward current from the set of traces (usually the peak inward current at 0 mV).
The pipette solution contained (in mM) 145 Cs+, 20 Cl, 2 EGTA, 5 HEPES, and 125 methanesulfonate (pH adjusted to 7.3 with CsOH). The bath solution contained (in mM) 10 Ba2+, 141.7 Na+, 4.7 K+, 166.4 Cl, 5 HEPES, and mannitol to reach a molarity of 290 mosM (pH adjusted to 7.35 with NaOH) for whole cell recordings. The 10 mM Ba2+ was used to increase the size of the current through L-type Ca2+ channels. All electrophysiological experiments were carried out at room temperature (2223°C).
NO Measurement
NO production was measured by using the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2) (2, 11, 16, 21). HEK-293 cells were passaged and plated onto 15-mm glass coverslips for transfection with the 1C- and
2-subunits of the L-type Ca2+ channel as described in Transfection of HEK-293 Cells with L-type Calcium Channel Subunits. At 48 h after transfection, the cells were washed twice and incubated for 10 min at 37°C with 10 µM DAF-2-DA (Sigma, St. Louis, MO) in OptiMEM (Invitrogen) supplemented with 0.1 mM L-arginine. The cells were washed again, resuspended in 1-ml OptiMEM, and viewed on an inverted epifluorescence microscope (model 5100TV; Zeiss, Germany) using light at 490 nm for excitation, and measuring the emitted light at 510 nm. Data were collected at 1-min intervals as 8-bit digital images using a SPOT camera (Diagnostic Instruments, Sterling Heights, MI). The fluorescence intensity was measured off-line for defined areas of interest in each image as grey-scale intensity with a custom macro written for the Zeiss KS400 software (Jim Tarara, Mayo Optical Morphology Core Facility, Rochester MN). Responses were quantified by measuring the initial slope of any observed change in fluorescence by linear regression (MS-Excel, Redmond, WA). The cells were preincubated with the NOS inhibitor N
-nitro-L-arginine (L-NAME, 1 mM) during DAF-2-DA loading to test the contribution of NOS to elevated NO levels. A positive control response was observed by adding bradykinin (1 µM) to the cells after loading (data not shown). CO was added from a 2% solution, to a final concentration of 0.2% as described in Chemicals and Data Analysis and the fluorescence was followed for 15 min. Specificity of DAF-2 fluorescence was established in preliminary experiments that demonstrated a linear relationship between DAF-2 fluorescence intensity in response to the calcium ionophore A23187
[GenBank]
and with parallel control experiments using DAF-4, a nonfluorescent analog (data not shown). In addition, we tested the response of DAF-2 (100 µM in intracellular solution) to a saturated solution of CO (
2.3% vol/vol) and observed no change in fluorescence, whereas addition of NO reproducibly caused a large increase in emitted light. There was no apparent interaction between CO and NO, because the DAF-2 response to NO was not affected when CO was added before or after NO (data not shown).
Chemicals and Data Analysis
The CO solution was prepared fresh before each experiment as previously described (36). Briefly, a gas bulb with a rubber injection port was filled with CO (Scott Specialty Gases, Troy, MI) at atmospheric pressure. A glass gas syringe was used to remove 1 ml of CO, which was added to 100 ml of bath solution placed in another glass gas bulb. One hundred microliters of 1% CO solution was gently added to the bath (500 µl) to prevent mechanoactivation of L-type Ca2+ channels (6) for a final CO concentration of 0.2%, 80 nM.
N-nitro-L-arginine (L-NNA), KT-5823, and KT-5720 were purchased from Sigma-Aldrich; 1H-[1,2,4]-oxadiazolo[4,3-]quinoxalin-1-one (ODQ), N-[3-(aminomethyl)benzyl] acetamidine·2HCl (1400W), and myristoylated PKC (2028) were from Biomol (Plymouth Meeting, PA). 3-Bromo-7-nitroindazole (3-Br-7-NI), N5-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO), and milrinone were from Tocris (Ellisville, MO).
L-NNA, ODQ, 3-Br-7-NI, KT-5823, and milrinone were dissolved in DMSO; 1400W was dissolved in water. The final concentration of DMSO did not exceed 0.1%. Control experiments with DMSO at this concentration showed no effect on the L-type Ca2+ current. All drugs were added to the external 10 mM Ba2+ Ringer solution and were incubated with the cells for at least 15 min before data were recorded.
Data are expressed as means ± SE. Values from the same cells before and after addition of CO or the drug of interest were evaluated by Student's t-test (two-tailed). A P value of <0.05 was considered significant.
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RESULTS |
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Effect of CO on L-type Ca2± channel current.
The cell capacitance for HEK cells was 19 ± 0.9 pF (n = 80), and the access resistance was 9.6 ± 0.6 M (n = 80). Peak Ba2+ current (IBa) typically increased during the first several minutes of recording before reaching steady state. After a minute at steady state, CO, at a final concentration of 0.2%, was added to the bath. Exogenous CO resulted in an increase in IBa recorded from expressed human intestinal Ca2+ channels in transfected HEK-293 cells (Fig. 1). CO (0.2%) increased IBa by 18 ± 3% (n = 21, P < 0.01); 19 of 21 cells showed an increase in IBa; in the other two cells, IBa was unchanged. There was no shift in the voltage dependence of the I-V relationship (Fig. 1). The increase in current evoked by CO was reversible on washout of CO (Fig. 1). The maximal solubility of CO in water is 2.3:100 ml, or by our nomenclature, 2.3%; 100 µl was added to a 500-µl bath resulting in a sixfold dilution. Therefore, the maximal concentration of CO that can be applied in this configuration is 0.38%. CO (0.38%) increased IBa by 21 ± 6% (n = 5, P < 0.005).
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Effect of inhibition of cGMP formation. CO activates guanylyl cyclase, resulting in an increase in cGMP levels (25, 41). To determine whether the observed effects of CO on the L-type Ca2+ current were through a cGMP pathway, we used ODQ to inhibit soluble guanylyl cyclase (12). Transfected HEK cells were pretreated with ODQ (10 µM) for 15 min, patch clamped, and after steady-state IBa was reached, CO (0.2%) was added to the bath. In the presence of ODQ, CO had no effect on IBa (CO changed IBa by 4 ± 3%; n = 14, P > 0.05, Fig. 2).
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Reduction in the stimulatory effects of CO on IBa NOS inhibition suggested that CO may be acting through the NOS/NO pathway. If that is the case, then NO donors should also increase IBa. Addition of the NO donor S-nitroso-N-acetylpenicillamine (SNAP; 20 µM) increased IBa by 24 ± 4% (n = 6, P < 0.05, Fig. 3).
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Human Jejunal Circular Smooth Muscle Cells
We also tested the effects of CO on the native L-type Ca2+ current in freshly dissociated human jejunal circular smooth muscle cells. Human jejunal circular smooth muscle cells had a capacitance of 71 ± 2 pF (n = 69). The access resistance was 7 ± 0.4 M (n = 69). Exogenous CO resulted in an increase in IBa in human jejunal circular smooth muscle cells by 14 ± 2% (n = 21, P < 0.01, Fig. 5) in standard whole cell recordings and by 12 ± 3% (n = 7, P < 0.05) in amphotericin B-perforated patch-clamp recordings. As with transfected HEK cells, no shift in the mean current-voltage relationships was noted, and the effects of CO were reversible on washing out CO from the bath solution. The soluble guanylyl cyclase inhibitor ODQ blocked the effects of 0.2% CO (3 ± 2% increase, n = 9, P > 0.05, Fig. 5). Similar to transfected HEK cells, 1400W (100 nM) did not completely block the stimulatory effects of CO (7 ± 1%, n = 6, P < 0.05), but in contrast to transfected HEK cells, 3 Br 7-NI (1 µM, 15 min) reduced the CO effect on IBa in human jejunal circular smooth muscle cells (5 ± 2%, n = 6, P > 0.5). L-NIO (5 µM, 15 min) blocked the CO effect of IBa in human jejunal circular smooth muscle cells (3 ± 1%, n = 6, P > 0.05), suggesting involvement of nNOS and possibly eNOS in the stimulatory effects of CO in human jejunal circular smooth muscle cells. The NO donor SNAP increased IBa by 14 ± 4% (n = 6 P < 0.05). We also tested the effects of protein kinase inhibitors on the stimulatory effect of CO on IBa. In the presence of the PKG inhibitor KT-5823 (1 µM, 15 min), CO increased IBa in human jejunal circular smooth muscle cells by a small but significant amount (5 ± 1%, n = 9, P < 0.05). KT-5720 (0.5 µM, 15 min), a PKA inhibitor, blocked the effects of CO on IBa (2 ± 2% increase, n = 7, P > 0.05). Milrinone (3 µM), the PDE III inhibitor, blocked the increase in IBa evoked by 0.2% CO (1 ± 1%, n = 10, P > 0.05).
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DISCUSSION |
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The relationship between CO and NO is complex (15). CO shares similar molecular weight and solubility characteristics with NO (9). As an endogenously produced gas, CO can activate guanylyl cyclase to produce cGMP, but its potency is considerably less than NO (41). The apparent disparity between the observed ability of CO to increase cGMP levels and the in vitro studies that suggest that CO is at best a weak direct activator of guanylyl cyclase has led to the suggestion that endogenous guanylyl cyclase may be sensitized to CO (10, 37). Cross talk between the NO and CO signaling pathways occurs at multiple levels. NO increases HO1 expression by inducing transcription and stabilizing HO mRNA (17) as well as through a cGMP-dependent pathway (32). CO regulates NOS activity in a concentration-dependent manner with high CO levels inhibiting NOS activity and low CO levels increasing NO production (40). CO is reported to induce directly release of NO from a cellular storage pool (40). Our data show that NOS inhibitors blocked the CO-induced activation of L-type Ca2+, suggesting that CO may not only induce release of preformed NO but also stimulates NO synthesis. The finding that CO can stimulate NO production provides another mechanism of action for CO and perhaps may explain the observed disparity between measured levels of cGMP induced by CO, and the known low affinity of guanylyl cyclase for CO as CO may indirectly increase cGMP levels through NO. The present studies suggest that CO can, in fact, activate the nNOS/NO/cGMP pathway, confirming that at low concentrations, CO is as an agonist of NO production. This is in contrast to a previously reported study in another species that suggested that CO had little effect on peak IBa in guinea pig ileal muscle (22). However, a much higher concentration of CO was used, which may have inhibited NO production.
The predominant mechanism of action of the effects of CO on L-type Ca2+ channels appeared to be through cAMP not cGMP. The direct effect of cGMP through PKG on L-type Ca2+ channels has been well studied and reviewed (20). The predominant effect of PKG activation by cGMP is inhibition of L-type Ca2+ channels (3, 19) either by direct or indirect phosphorylation of the -subunit or through increased production of PDE II, resulting in increased breakdown of cAMP and, therefore, reduced PKA levels. It is possible that CO had a dual effect on L-type Ca2+ channels in human intestinal smooth muscle, inhibiting channel activity through cGMP and increasing channel activity through cAMP. However, inhibition of PKG did not further increase the stimulatory effects of CO on native and expressed human intestinal L-type Ca2+ channels over the stimulation seen in the absence of PKG inhibitors, suggesting that the cGMP/PKG pathway was not a major regulatory pathway for the effects of CO on L-type Ca2+ channels in either of these cell types. In contrast, inhibition of PKA did block the effects of CO on the L-type Ca2+ channels implicating this pathway in CO stimulation of L-type Ca2+ channels. Cross talk exists between the PKG and PKA pathways (reviewed in Ref. 20). Elevated cGMP levels can activate directly PKA as both cGMP and cAMP are not absolutely specific for their respective kinases. cGMP also inhibits PDE III. Inhibition of PDE III results in decreased breakdown of cAMP (33), resulting in an elevation of endogenous cAMP levels and further stimulation of PKA. The effects of PKA on intestinal smooth muscle L-type Ca2+ channels has been reported predominantly, but not exclusively, to be stimulatory, similar to its effects in cardiac myocytes. However, PKA stimulation of intestinal L-type Ca2+ channels is reported to be less pronounced than PKA stimulation of cardiac myocytes and does not left-shift the I-V relationship (reviewed in Ref. 20). Our findings are also in agreement with these observations, with a modest increase in IBa and no shift in the I-V relationships.
Whether endogenously produced CO regulates L-type Ca2+ channels in intestinal smooth muscle is not known. Our experiments utilized low concentrations of exogenous CO. The major source of endogenous CO is through the heme breakdown pathway catalyzed by heme oxygenase. Under unstimulated conditions, HO2 is the predominant isoform expressed in the gastrointestinal tract (26). HO1 is expressed ubiquitously in mammalian cells and is upregulated in response to a large variety of stimuli, including inflammation (26). HO2 is expressed predominantly in enteric nerves, interstitial cells of Cajal, and mucosal epithelial cells of mouse (27). Endogenously produced CO can modulate neurotransmission and act as a hyperpolarizing factor (43), suggesting that enough CO is produced to have a physiological effect. However, the microenvironment around the L-type Ca2+ channel will determine whether CO also functions as a regulator of L-type Ca2+ channels in vivo as well as a modulator of neurotransmission, inflammation, Ca2+ sparks, and as a hyperpolarizing factor.
In summary, our data suggest that low levels of exogenous CO can activate native and heterologously expressed intestinal L-type Ca2+ channels through a pathway that involves activation of NOS, increased NO levels, and increased cGMP but does not appear to involve PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of PDE III. CO-induced NO production may explain the apparent discrepancy between the low affinity of guanylyl cyclase for CO and the robust cGMP production evoked by CO.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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