The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (KCa) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K+ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance KCa channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single KCa channels in SMCs. Furthermore, the effect of CO on KCa channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, Gi/Go or Gs) in excised membrane patches. Our results suggest that the direct modulation of high-conductance KCa channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO.

The chemical modification of big conductance calcium-activated potassium (K Ca ) channels in rat tail artery smooth muscle cells by carbon monoxide (CO) was investigated using the cell-free single channel recording technique. Exposure of the internal surface of cell membranes to diethyl pyrocarbonate (DEPC) neither affected the characteristics of K Ca channels nor modified the stimulatory effect of CO on K Ca channels. However, when DEPC was applied to the external surface of cell membranes, the open probability of K Ca channels was reduced. The pH and concentration dependence of the effect of DEPC indicated the specific modification of histidine residues. Kinetic analysis suggested that one externally located histidine residue was modified by DEPC. Treatment of the external surface of cell membranes with DEPC abolished the CO-induced increase in the open probability of K Ca channels. Likewise, the presence of CO partially protected K Ca channels from inhibition by DEPC. Moreover, photooxidation of the histidine residue located on the external membrane surface abolished the CO-induced activation of K Ca channels. Our study demonstrates that the CO-induced increase in the open probability of K Ca channels may rely specifically on the structure and topological locations of histidine residues.The presence of both inducible and constitutive forms of heme oxygenase, which cleave the heme ring to form carbon monoxide (CO), 1 has been demonstrated in vascular smooth muscle cells (1). Direct measurement of the endogenous production of CO from vascular smooth muscle cells has also recently been realized (2). Our previous study (3), as well as those of others (4 -6), demonstrated the regulatory function of CO in various types of vascular tissues. For example, CO relaxed precontracted rat tail artery strips in a concentration-dependent manner (3). This effect of CO was mediated partially by a cGMP signaling pathway and partially by calcium-activated K (K Ca ) channels. Using the single channel recording patch-clamp technique, we found that CO increased the open probability (NPo) of K Ca channels. 2 This effect of CO may be caused by a direct interaction between CO and K Ca channels as the activities of the cGMP pathway and G proteins are not involved in the activation of K Ca channels by CO. To date, the mechanism by which CO directly affects K Ca channels remains unclear.The electrical properties of K Ca channels are determined by different amino acid residues that are constitutive components of the channel protein. The primary structure of K Ca channels in several cell types, excluding vascular smooth muscle cells, is known, but the functional roles of various amino acids in the gating and conducting of K Ca channels are still in question. Limited studies show that the modification of one or more amino acid residues may significantly change the conductance and/or NPo of K Ca channels (7-9). Both synthetic chemical reagents and some simple biological active molecules such as nitric oxide (7) and hydrogen peroxide (10) can...

1 Carbon monoxide (CO) induced a concentration-dependent relaxation of isolated rat tail artery tissues which were precontracted with phenylephrine or U-46619. This vasorelaxing e ect of CO was independent of the presence of the intact endothelium. 2 The CO-induced vasorelaxation was partially inhibited by the blockade of either the cyclicGMP pathway or big-conductance calcium-activated K (K Ca ) channels. When both the cyclicGMP pathway and K Ca channels were blocked, the CO-induced vasorelaxation was completely abolished. 3 Incubation of vascular tissues with hemin, in order to enhance the endogenous production of CO, suppressed the phenylephrine-induced vasocontraction in a time-and concentration-dependent manner. The hemin-induced suppression of the vascular contractile response to phenylephrine was abolished after the vascular tissues were co-incubated with either oxyhaemoglobin or zinc protoporphyrin-IX, suggesting an induced endogenous generation of CO from vascular tissues.4 The e ect of hemin incubation on vascular contractility did not involve the endogenous generation of nitric oxide. 5 Our results suggest that CO may activate both a cyclicGMP signalling pathway and K Ca channels in the same vascular tissues, and that the endogenously generated CO may signi®cantly a ect the vascular contractile responses.

An enhanced .O2- formation resulting from an increased NADH oxidase activity was found in aorta from SHR and DOCA-HT rats. Cultured arterial SMCs from SHR also generated excessive .O2- formation under basal and stimulated conditions. The age-related increase in vascular .O2- formation in association with the rise in blood pressure in SHR suggests that the oxidative stress might contribute to the development of hypertension. NADH oxidase activity was greater in aorta of both hypertension models, but a decrease of Cu/Zn SOD activity could also contribute to the high level of aortic .O2- in DOCA-HT rats.

Several experimental and clinical evidences have linked an enhanced production of reactive oxygen species (ROS) to certain diseases of the cardiovascular system including hypertension and diabetes. However, it has never been clearly established whether the enhanced oxidative stress observed in those conditions is primary or secondary to the pathological process. Our experimental studies have permitted to demonstrate that ROS, mainly through the production of superoxide anion, can cause important alterations in the cellular signal transduction systems characterized by an enhanced production of inositol triphosphate and a reduced production of cyclic GMP in cultured vascular smooth muscle cells (SMC), thus favouring the vasoconstriction. Since those effects were found to be increased in SMC from spontaneously hypertensive rats (SHR), this suggested a greater sensitivity of the vascular tissue of SHR to the oxidative stress. Moreover, we also have observed an increased production of superoxide anion in the aorta of rats made hypertensive according to the SHR, glucose or angiotensin-induced and DOCA-salt models during the development of hypertension. Since the superoxide anion production could be correlated with the level of blood pressure and since the development of hypertension could be either totally prevented or markedly attenuated by chronic treatment with potent antioxidative therapies such as alpha lipoic acid or aspirin, this suggested a major contribution of vascular superoxide anion production in the development of hypertension in those models. Moreover, the development of insulin resistance, which is associated to the model of glucose-induced hypertension, was also found to be prevented by chronic antioxidant therapies, thus suggesting that oxidative stress plays an important role as well in the development of insulin resistance and type 2 diabetes. In conclusion, it appears that oxidative stress may constitute a major pathogenic factor in the development of hypertension and type 2 diabetes. Moreover, our studies suggest that the chronic treatment with appropriate antioxidative therapies could prevent the development of hypertension and diabetes as well as their complications in various experimental models of hypertension.

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Using a microfluidics device, fluorescence of a recorder (mCherry or mVenus) gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously on over 1000 single cells of Neurospora crassa every half hour for 10 days under each of varied light and temperature conditions. Single cells were able to entrain to light over a wide range of day lengths, including 6, 12, or 36 h days. In addition, the period of oscillations in fluorescence remained remarkably stable over a physiological range of temperatures from 20 o C to 30 o C (Q 10 = 1.00-1.07). These results provide evidence of an autonomous clock in most single cells of N. crassa. While most cells had clocks, there was substantial variation between clocks as measured by their phase, raising the question of how such cellular clocks in single cells phase-synchronize to achieve circadian behavior in eukaryotic systems at the macroscopic level of 10 7 cells, where most measurements on the clock are performed. Single cells were placed out of phase by allowing one population to receive 6 or 12 h more light before lights out (D/D). The average phase difference was reduced in the mixed population relative to two unmixed control populations.