In order to elucidate the signal transduction pathways of vascular smooth muscle contractions induced by stimulation of receptors for 5-hydroxytryptamine (5-HT) and thromboxane A2 (TXA2), both of which are released from activated platelets, we examined whether protein kinases, such as tyrosine kinase, p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC), are involved in the contraction produced by either 5-HT or U46619 (an analog of TXA2) in the rat aorta. Both 5-HT and U46619 induced sustained contractions, which were markedly reduced in the absence of extracellular Ca2+. Verapamil (a L-type Ca2+ channel blocker) markedly inhibited the contractile response to 5-HT, while the U46619-induced contraction was only slightly inhibited by verapamil. Both contractile responses to 5-HT and U46619 were significantly inhibited by calphostin C (a PKC inhibitor). On the other hand, both genistein (5 μM, a tyrosine kinase inhibitor) and SB203580 (a p38 MAPK inhibitor) significantly inhibited 5-HT-induced contractions but had little effects on the contractions induced by U46619. These results suggest that the signal transduction mechanisms involved in the contractions mediated via 5-HT and TXA2 receptors are different as follows. Both the tyrosine kinase and p38 MAPK pathways are involved in 5-HT contraction but not in TXA2 contraction, while both contractions are strongly dependent on transplasmalemmal Ca2+ entry. The contractile responses to both 5-HT and TXA2 involve voltage-dependent Ca2+ channels and PKC.
The electrophysiological properties of smooth muscle in the murine lower oesophageal sphincter (LOS) were investigated by intracellular microelectrode recording. Inhibitory junction potentials (IJPs) evoked by trains of field stimulation (30 V, 0.2-0.3 ms, 10 stimuli at 1-50 Hz) were observed in the murine LOS in the presence of atropine (1 μM) and nifedipine (1 μM). The IJP consists of two components, which we termed fast IJP and slow IJP. The fast IJP was partly sensitive to guanethidine (5 μM), pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 μM) and apamin (0.1 μM), suggesting that the fast IJP was produced partly through the activation of apamin-sensitive Ca2+-activated K+ channels and of P2-purinoceptors. The other part of the fast IJP was sensitive to Nω-nitro-l-arginine (l-NNA, 100 μM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 1 μM), but insensitive to apamin (0.1 μM), iberiotoxin (50 nM) and charybdotoxin (30 nM). Slow IJP was sensitive to l-NNA (100 μM), ODQ (10 μM) and glibenclamide (10 μM), but insensitive to apamin (0.1 μM), iberiotoxin (50 nM) and charybdotoxin (30 nM). KT5823, a protein kinase G (PKG) inhibitor, had no effect on the fast and slow IJP in this tissue. It was suggested that, in the mouse LOS, adenosine trisphosphate (ATP) partly mediated the fast IJP through apamin-sensitive Ca2+-activated K+ channels, and nitric oxide mediated the remained part of the fast IJP and the slow IJP through cGMP, but not PKG. ATP-sensitive K+ channels were suggested to be partly involved in the production of slow IJP, but the responsible channel(s) for the nitrergic fast IJP remained unclarified.
This review will focus on the pacemaker mechanisms underlying gastrointestinal autonomic rhythmicity in an attempt to elucidate the differences and similarities between the pacemaker mechanisms in the heart and gut. Interstitial cells of Cajal (ICC) form networks that are widely distributed within the submucosal (ICC-SM), intra-muscular (ICC-IM, ICC-DMP) and inter-muscular layers (ICC-MY) of the gastrointestinal tract from the esophagus to the internal anal sphincter. The ICC generate spontaneously active pacemaker currents that may be recorded as plateau and slow potentials. These pacemaker currents drive the spontaneous electrical and mechanical activities of smooth muscle cells. The enteric nervous system, composed of both the myenteric (inter-muscular) plexus and the submucosal plexus, is also distributed in the gastrointestinal tract from the esophagus to the internal anal sphincter. The role of the ICC and the enteric nervous system in the integrative control of gastrointestinal function and especially of spontaneous rhythmic activity, is still unknown. Nevertheless, at least from the results presented in this review of studies of the jejunum, ileum and proximal colon of the mouse, it is convincing that the ICC drive spontaneous rhythmic motility, although a role for the enteric nervous system in the regulation of spontaneous rhythmic motility cannot be overlooked. Furthermore, intracellular Ca2+ handling has a critical role in the generation of pacemaker activity in the gut and heart, although respective players such as the Ca2+-ATPase of the sarcoplasmic reticulum (endoplasmic reticulum), IP3 receptors, ryanodine receptors and plasma membrane ion channels may have divergent roles in the Ca2+-release refilling cycles. In conclusion, intracellular Ca2+ handling plays a key role in the gut pacemaker responsible for spontaneous rhythmicity, as well as in the cardiac pacemaker responsible for spontaneous beating. Pharmacotherapeutic targeting of intracellular Ca2+ handling mechanisms may be a promising approach to the treatment and cure of gut motility dysfunction.
Electrophysiological properties of pacemaker potentials recorded from myenteric interstitial cells of Cajal (ICC-MY) within the guinea-pig gastric antrum are reviewed briefly. Pacemaker potentials consist of two components, a primary component forming a transient depolarization with a rapidly rising initial phase, followed by a secondary component as a plateau with sustained depolarization. The primary component is inhibited by low [Ca2+]o solutions or depolarization of the membrane with high [K+]o solutions. This inhibition could be mimicked by chelating [Ca2+] i with BAPTA-AM, suggesting that this component is produced by activation of voltage-dependent Ca2+ permeable channels. The plateau component is inhibited by low [Cl-] o solution or DIDS, an inhibitor of Ca2+-activated Cl--channels, suggesting that this component is formed by Ca2+-activated Cl--currents. Reduction of Ca2+ release from internal stores by inhibiting the internal Ca-pump with cyclopiazonic acid results in a shortened duration of the plateau component, with no alteration in the rate of rise of the primary component. 2-APB, an inhibitor of the IP3-receptor mediated Ca2+ release from internal stores, abolishes pacemaker potentials, suggesting that the release of Ca2+ from internal IP3-sensitive Ca2+ stores is required for generation of pacemaker potentials. CCCP, a mitochondrial protonophore, depolarizes the membrane and abolishes pacemaker potentials, suggesting that mitochondrial Ca2+ handling functions may be coupled with generation of pacemaker potentials. These results indicate that the two components of pacemaker potentials are generated by different mechanisms; the primary component may be produced by activation of voltage-dependent Ca2+-permeable channels, while the plateau component may be produced by the opening of Ca2+-activated Cl--channels. It is hypothesized that pacemaker potentials are initiated by depolarization of the membrane due to generation of unitary potentials in response to mitochondrial Ca2+ handling. Activation of voltage-dependent Ca2+ influx, IP3-receptor mediated Ca2+ release from the internal stores and Ca2+-activated Cl--channels may be involved as successive steps downstream to the generation of unitary potentials.
The cardiac pacemaker is a sino-atrial (SA) nodal cell. The signal induced by this pacemaker is distributed over the heart surface by a specialised conduction system and is clinically recorded as the ECG. The SA nodal cells are highly resistant to cardiac failure and ischemia. Under calcium overload conditions, some dysrythmias of SA nodal cells occur easily. Morphological analysis under these conditions shows swelling of the cisternae of the Golgi apparatus, with little or no other histological change or damage being observed. The rate of sinus rhythm is quite different between various species. The investigations of SA nodal cells have so far clarified the pacemaker mechanisms involved. A number of ionic channel currents or pacemaker currents, contribute to the depolarization of the pacemaker potential (phase 4). This will not occur with a single current. Recent experiments have identified several novel pacemaker currents and have also revealed several differences in the pacemaker currents between species. The marked hyperpolarization-activated inward current (If) appears in SA nodal cells of most species, while the inwardly rectifying K+ current (IK1) with masked If current is found in those of the rat and monkey. In addition, the rapidly activated current (IKr) and slowly activated current (IKs) of the delayed rectifier K+ current (IK) contribute to the pacemaker potential in guinea pig SA nodal cells, with only the IKs current in porcine SA nodal cells and only the IKr current in the rat and rabbit. These differences in ionic channels presumably result from differences in gene expression. Some smooth muscle cells also possess the capacity to beat spontaneously. Uterine smooth muscle cells also exhibit an If current. The basal mechanism for spontaneous activity in both SA nodal cells and smooth muscle cells is almost the same, but some differences in the ionic channels and their genetic expression may contribute to their respective pacemaker currents.
In cardiac sino-atrial node (SAN) cells, time- and voltage-dependent changes in the gating of various ionic currents provide spontaneous, stable and repetitive firing of action potentials. To address the ionic nature of the species-dependent heart rate, action potentials and membrane currents were recorded in single cells dissociated from the porcine SAN, and compared with those from SAN cells of rabbits, guinea-pigs and mice. The porcine SAN cells exhibited spontaneous activity with a frequency of 60-80 min-1, which was much slower than that of rabbit SAN cells. Under voltage clamp conditions, depolarization activated the L-type Ca2+ current (ICaL) followed by a gradual activation of the delayed rectifier K+ current (IK) while hyperpolarization activated the hyperpolarization-activated cation current (Ih). It was found that the major component of IK in porcine SAN is the slowly activating IK (IKs), in contrast to SAN cells of the rabbit and other species in which the rapid IK (IKr) plays an active role in repolarization and the subsequent pacemaker depolarization. Replacement of rabbit IKr with porcine IKs and a slight modification in the gating parameters and amplitudes of other current systems in the `Kyoto Model' gave an adequate reconstruction of spontaneous action potentials as well as of the voltage clamp recordings. We conclude that the density and the kinetics of IK contribute, in part, to the different heart rates of various species.