CARBON MONOXIDE

Carbon monoxide is a gas that is physiologically produced in most species including humans. The major route of production of carbon monoxide is through the breakdown of heme by heme oxygenase. Three isoforms of heme oxygenase have been described. Work in the laboratory has described activation of a delayed rectifier potassium channel by carbon monoxide, and the presence of heme oxygenase in a subset of enteric neurons and in murine interstitial cells of Cajal. Recent work on knockout mice lacking heme oxygenase-2 has revealed a role for carbon monoxide as a novel inhibitory neurotransmitter in the gastrointestinal tract.

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Figure 1.1:

Activation of a potassium current by CO.

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Figure 1.2:

Immunofluorescent images obtained from six sequential optical sections taken at the level of the IC-MP showing immunopositive structures for c-Kit and HO-2. The c-Kit field in A shows at least three immunopositive IC cell bodies. The image in B shows that the same three IC bodies were also immunopositive for HO-2. The combined c-Kit and HO-2 fields are shown in C. Note the colocalization of c-Kit-like and HO-2-like immunoreactivities in the IC bodies, whereas only weak HO-2 signal was observed in the IC processes. Bar = 50 µm.

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Figure 2.1.

Targeted genomic deletion of HO2 and nNOS results in depolarization of the RMP of jejunal circular smooth muscle cells. The RMP of smooth muscle cells from HO2 ^{^/^} , nNOS ^{^/^} , HO2 ^{^/^} ynNOS ^{^/^} , and wild-type (WT) mice was determined as described in Materials and Methods. HO2 ^{^/^} smooth muscle cells are depolarized compared with wild-type cells with RMPs of 257.1 6 0.5 mV (n 5 115) and 265.4 6 0.4 mV (n 5 118; p, P , 0.001), respectively. nNOS ^{^/^} smooth muscle cells are also depolarized with respect to wild-type cells with an RMP of 259.2 6 0.7 mV (n 5 45; p, P , 0.05). HO2 ^{^/^} ynNOS ^{^/^} cells are even more depolarized with an RMP of 251.8 6 0.7 mV (n 5 55; p, P , 0.01). As shown, tetrodotoxin (TTX) had no effect on the RMP with an RMP of 263.5 6 3mV (n 5 5) in the absence of tetrodotoxin and of 262.3 6 2 mV (n 5 5; P . 0.05) in the presence of tetrodotoxin (500 uM).

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Figure 2.2.

HO2 is required for normal inhibitory neurotransmission in intestinal smooth muscle. (A) Targeted genomic deletion of HO2 results in attenuated mechanical and electrical responses to EFS. Mechanical (Upper traces) and intracellular electrical (Lower traces) activities were recorded from jejunal circular smooth muscle cells derived from wild-type (WT) and HO2 ^{^/^} mice. EFS (six pulses each of 0.35 ms, 100 V, and 30 Hz) was applied as indicated by the asterisk (*). Under NANC conditions, EFS elicited a hyperpolarization, an inhibitory junctional potential (IJP), with an amplitude of 7.4 60.4 mV(n 526 measurements; 17 preparations) in wild-type specimens. Inhibition of electrical slow wave activity and contractile activity follows the EFS-evoked IJP in wild-type specimens with recovery of activity in 15?25 s. In HO2 ^{^/^} ?derived samples, EFS evokes an initial IJP of only 2.6 6 0.4 mV (n 5 110 cells; 22 preparations; P ,0.05) with an attenuation in the reduction of electrical slow waves. Moreover, EFS did not abolish, but only modestly decreased, contractile activity in HO2 ^{^/^} -derived muscle strips with a mean decrease in contractile activity of 12 6 3% (n 5 10 preparations). Recovery of contractile activity was significantly faster than in wild-type specimens, with full recovery in less than 10 s. (B) Graph of the most hyperpolarized portion of each electrical slow wave before, during, and after EFS. The voltage corresponding to the most negative potential for each electrical slow wave was plotted before, during, and after EFS. EFS (six pulses each of 0.35 ms, 100 V, and 30 Hz) was given at the time indicated (see asterisk). The RMP is depolarized in the HO2 ^{^/^} -derived speci-men compared with the wild type.

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Figure 2.3.

Targeted genomic deletion of nNOS, HO2, or both genes attenuates electrical and mechanical responses to EFS. Mechanical (Upper) and electrical(Lower) response to EFS (*) recorded from jejunal circular smooth muscle cells from wild-type (WT), nNOS ^{^/^} , and HO2 ^{^/^} ynNOS ^{^/^} mice. EFS evokes an IJP of only 20.75 60.8 mVin nNOS ^{^/^} -derived specimens compared with 27.6 60.6 mVin wild-type muscles (n 54; P ,0.05). However, the decrease in slow wave amplitude was not different between nNOS ^{^/^} and wild-type specimens, with a decreased amplitude of 45 65%and 47 63%, respectively (n 512; P .0.05). Both electrical and mechanical inhibitory responses to EFS were nearly abolished in the HO2 ^{^/^} ynNOS ^{^/^} -derived samples, and an excitatory electrical response was unmasked.

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Table 2.1.

Targeted genomic deletion of HO2 attenuates the electrical response to EFS.

Targeted genomic deletion of HO2 results in attenuation of electrical response to EFS in jejunal smooth muscle preparations. The initial hyperpolarization or IJP in response to EFS is only 2.6 mV in HO2 ^{^/^} -derived specimens compared to 7.6 mV in wild-type samples. In addition, the reduction in the amplitude of the first three electrical slow waves after EFS is significantly attenuated in the HO2 ^{^/^} -derived specimens compared to wild-type samples. The number of independent measurements is indicated by (n), and statistical significance is indicated (*, P <0.05).

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Figure 2.4.

Exogenous CO restores NANC inhibitory transmission in HO2 ^{^/^} jejunal smooth muscles. Mechanical and electrical responses to EFS were monitored in HO2 ^{^/^} and HO2 ^{^/^} ynNOS ^{^/^} -derived jejunal smooth muscles as indicated. In some cases (B?D), the perfusing buffer was treated with CO (10% volyvol) for 10 min before the experiment. In one case (C), the buffer contained L-nitroarginine (L-NNA; 100 mM) for 20 min before EFS. The mean hyperpolarization evoked by CO was 23.6 60.7 mV(n 55) and 23.0 60.6 mV (n 58) in wild-type and HO2 ^{^/^} -derived preparations, respectively, indicating HO2 ^{^/^} -derived smooth muscle retains the ability to respond to exogenous CO. (A) EFS evokes only a small IJP and a modest reduction in mechanical activity from HO2 ^{^/^} -derived muscles (see also Fig. 2 A and B). (B) After perfusion with CO, the inhibition of mechanical activity in response to EFS is restored, although the amplitude of the EFS-evoked IJP remains small. (C and D) Interestingly, the increase in EFS-evoked inhibition of contractile-mechanical activity after COapplication is abolished in the presence of 100 mM L-nitroarginine (C) and is absent in HO2 ^{^/^} ynNOS ^{^/^} -derived specimens (D). Thus, the effect of exogenous CO is not apparent when nNOS is absent (HO2 ^{^/^} ynNOS ^{^/^} muscles) or when NO production is blocked (by treatment with L-nitroarginine), indicating that CO may require NO for its physiologic effects.

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CARBON MONOXIDE ACTIVATES HUMAN INTESTINAL SMOOTH MUSCLE L-TYPE Ca2+ CHANNELS THROUGH A NITRIC OXIDE-DEPENDENT MECHANISM

Figure 3.1.

Effect of exogenous carbon monoxide (CO) on the L-type Ca2+ channel currents in transfected HEK-293 cells. A: inward currents were recorded from HEK-293 cells transfected with human intestinal smooth muscle alpha-1C (CaV1.2)- and beta-2-subunits. CO (0.2%) increased maximal peak Ba2+ current (IBa) by 18 ± 3% (n = 21, P < 0.01). B: normalized current-voltage relationships are shown [control transfected HEK-293 cells (triangle), CO (diamond)]. C: normalized IBa before and during CO exposure and after removal of CO from the bath (*P < 0.05).

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Figure 3.2.

Block of the effect of CO after pretreatment with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). A: in the presence of ODQ (10 uM, 15 min), exposure to CO (0.2%) had no effect on IBa in HEK-293 cells transfected with the human jejunal alpha-1C-and beta-2-subunits. B: mean current voltage relationships [transfected HEK-293 cells (triangle), CO (diamond)] and the normalized IBa.

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Figure 3.3.

Increase in L-type Ca2+ channel current induced by the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP). A: NO donor SNAP increased IBa in transfected HEK-293 cells (10 uM). B: mean current voltage relationships [transfected HEK-293 cells (triangle), with SNAP (diamond)]. C: normalized IBa. (*P < 0.05).

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Figure 3.4.

CO activates NO production in HEK-293 cells. Effect of CO on 4,5-diaminofluorescein (DAF-2) fluorescence in HEK-293 cells transfected with the alpha-1C- and beta-2-subunits of the L-type Ca2+ channel. A: increase in DAF-2 fluorescence with time [0.2% CO circle, 0.2% CO and Nw-nitro-L-arginine (L-NAME) square]. B: mean slope of fluorescence response to CO. CO (0.2%) was added 5 min before the initial data point. Cells were incubated with L-NAME (1 mM) for 10 min during DAF-2 loading to inhibit NOS activity. Data are means ± SE for 23 cells (*P < 0.05).

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Figure 3.5.

Effect of exogenous CO on the L-type Ca2+ channel currents in human jejunal circular smooth muscle cells. A: IBa were recorded from freshly dissociated human jejunal circular smooth muscle cells. CO (0.2%) increased IBa by 14 ± 2% (n = 21, P < 0.01). B: current voltage relationships (control circle, CO square). C: mean maximal response. In the presence of ODQ (10 uM, 15 min), exposure to CO (0.2%) had no effect on IBa (D), whereas the NO donor SNAP increased IBa (E).

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Selected Publications (Complete list of publications)

• Perri RE, Langer DA, Chatterjee S, Gibbons SJ, Gadgil J, Cao S, Farrugia G, Shah VH. Defects in cGMP-PKG pathway contribute to impaired NO dependent responses in hepatic stellate cells upon activation. Am J Physiol Gastrointest Liver Physiol. 2005 Nov 3; [Epub ahead of print]
• Lim I, Gibbons SJ, Lyford GL, Miller SM, Strege PR, Sarr MG, Chatterjee S, Szurszewski JH, Shah VH, Farrugia G. Carbon monoxide activates human intestinal smooth muscle L-type Ca2+ channels through a nitric oxide-dependent mechanism. Am J Physiol Gastrointest Liver Physiol. 2005 Jan;288(1):G7-G14.
• Szurszewski JH, Farrugia G. Carbon monoxide is an endogenous hyperpolarizing factor in the gastrointestinal tract. Neurogastroenterol Motil. 2004 Apr;16 Suppl 1:81-5.
  
• Shah V, Lyford G, Gores G, Farrugia G. Nitric oxide in gastrointestinal health and disease. Gastroenterology. 2004 Mar;126(3):903-13.
  
• Gibbons SJ, Farrugia G. The role of carbon monoxide in the gastrointestinal tract. J Physiol. 2004 Apr 15;556(Pt 2):325-36. Epub 2004 Feb 06.
  
• Gonzalez-Michaca L, Farrugia G, Croatt AJ, Alam J, Nath KA. Heme: a determinant of life and death in renal tubular epithelial cells. Am J Physiol Renal Physiol. 2004 Feb;286(2):F370-7.
  
• Farrugia G, Lei S, Lin X, Miller SM, Nath KA, Ferris CD, Levitt M, Szurszewski JH. A major role for carbon monoxide as an endogenous hyperpolarizing factor in the gastrointestinal tract. Proc Natl Acad Sci U S A. 2003 Jul 8;100(14):8567-70. Epub 2003 Jun 27.
  
• Zyromski NJ, Duenes JA, Kendrick ML, Balsiger BM, Farrugia G, Sarr MG. Mechanisms mediating nitric oxide induced inhibition in human jejunal longitudinal smooth muscle. Surgery 2001 Sep; 130(3):489-96.
  
• He C-L, Soffer EE, Ferris CD, Walsh RM, Szurszewski JH, Farrugia G. Loss of interstitial cells of Cajal and inhibitory innervation in insulin-dependent diabetes Gastroenterology 2001 Aug; 121(2): 427-34.
  
• Miller SM, Reed D, Sarr MG, Farrugia G, Szurszewski JH. Heme oxygenase in enteric nervous system of human stomach and jejunum and co-localization with nitric oxide synthase Neurogastroenterology and Motility 13 (2):121-131, 2001.
  
• Xue L, Farrugia G, Miller SM, Ferris CD, Snyder SH, Szurszewski JH. (2000). Carbon monoxide and nitric oxide as co-neurotransmitters in the enteric nervous system: evidence from genomic deletion of biosynthesis enzymes. Proc. Natl. Acad. Sci. 97:1851-1855.
  
• Farrugia G, Szurszewski JH. (1999). Heme oxygenase, carbon monoxide, and interstitial cells of Cajal. Microscopy Research and Techniques. 47(5):321-4.
  
• Farrugia G, et al. (1998). Distribution of heme oxygenase and effects of exogenous carbon monoxide in canine jejunum. Am J Physiol. 274(2 Pt 1):G350-8.
  
• Rich A, et al. (1994). Carbon monoxide stimulates a potassium-selective current in rabbit corneal epithelial cells. Am J Physiol. 267(2 Pt 1):C435-42.
  
• Farrugia G, et al. (1993). Activation of whole cell currents in isolated human jejunal circular smooth muscle cells by carbon monoxide. Am J Physiol. 264(6 Pt 1):G1184-9.
  
• Sha L, Farrugia G, Harmsen WS, Szurszewski JH. Membrane potential gradient is carbon monoxide-dependent in mouse and human small intestine. Am J Physiol Gastrointest Liver Physiol. 2007 Aug;293(2):G438-45.
  
• Miller SM, et al. (1997). Heme oxygenase 2 is present in interstitial cell networks of the mouse small intestine. Gastroenterology. 114(2):239-44.