Role of Ba2+-resistant K+ channels in endothelium-dependent hyperpolarization of rat small mesenteric arteries

In rat small mesenteric arteries, the influence of modulation of basal smooth muscle K+ efflux on the mechanism of endothelium-dependent hyperpolarization was investigated. The membrane potentials of the vascular smooth muscle cells were measured using conventional microelectrode techniques. Incubation of resting arteries with the gap junction uncoupler carbenoxolone (20 micro M) decreased the endothelium-dependent hyperpolarization elicited by a submaximal concentration of acetylcholine (3 micro M) to about 65% of the control. In the presence of Ba2+ (200 micro M), which depolarized the membrane potential by 10 mV, the acetylcholine-induced membrane potential response was doubled in magnitude, reaching values not different from control. Moreover, the hyperpolarization was more resistant to carbenoxolone in these conditions. Finally, both in the absence and in the presence of carbenoxolone, the combined application of Ba2+ and ouabain (0.5 mM) did not abolish the acetylcholine response. These results suggest that gap junctional coupling plays a role in endothelium-dependent hyperpolarization of smooth muscle cells of resting rat small mesenteric arteries. Additionally, these findings show that the hyperpolarization does not rely on activation of inward rectifying K+ channels. Although a minor contribution of Na-K pumping cannot be excluded, the Ba2+ experiments show that the membrane electrical response is mediated by activation of a Ba2+-resistant K+ conductance.     

Local inhibitory reflexes excited by mucosal application of nutrient amino acids in guinea pig jejunum

The motility of the gut depends on the chemicals contained in the lumen, but the stimuli that modify motility and their relationship to enteric neural pathways are unclear. This study examined local inhibitory reflexes activated by various chemical stimulants applied to the mucosa to characterize effective physiological stimuli and the pathways they excite. Segments of the jejunum were dissected to allow access to the circular muscle on one-half of the preparation while leaving the mucosa intact on the circumferentially adjacent half. Chemicals were transiently applied to the mucosa, and responses were recorded intracellularly in nearby circular muscle cells. The amino acids l-phenylalanine, l-alanine, or l-tryptophan (all 1 mM) evoked inhibitory junction potentials (IJPs; latency 150-300 ms, amplitude 3-8 mV, each n > 6) that were blocked by TTX and partially blocked by antagonists of P2X receptors and/or a combination of antagonists at 5-HT(3) and 5-HT(4) receptors. The putative mediators 5-HT (10 microM), ATP (1 mM), and CCK-8 (1-10 microM) elicited IJPs mediated via 5-HT(3), P2X, and CCK-B receptors, respectively. Responses were only partially reduced by the effective antagonists. IJPs evoked by electrically stimulating the mucosa were unaffected by antagonists that reduced chemically evoked responses. Both chemically and electrically evoked IJPs were resistant to nicotinic, NK(1), NK(3), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-d-aspartate, or CGRP receptor blockade. We conclude that mucosal stimulation by amino acids activates local neural pathways whose pharmacology depends on the nature of the stimulus. Transmitters involved at some synapses in these pathways remain to be identified.     

Inhibitory neuromuscular transmission mediated by the P2Y1 purinergic receptor in guinea pig small intestine

ATP is a putative inhibitory neurotransmitter responsible for inhibitory junction potentials (IJPs) at neuromuscular junctions (IJPs) in the intestine. This study tested the hypothesis that the purinergic P2Y(1) receptor subtype mediates the IJPs. IJPs were evoked by focal electrical stimulation in the myenteric plexus and recorded with "sharp" intracellular microelectrodes in the circular muscle coat. Stimulation evoked three categories of IJPs: 1) purely purinergic IJPs, 2) partially purinergic IJPs, and 3) nonpurinergic IJPs. Purely purinergic IJPs were suppressed by the selective P2Y(1) purinergic receptor antagonist MRS2179. Purely purinergic IJPs comprised 26% of the IJPs. Partially purinergic IJPs (72% of the IJPs) consisted of a component that was abolished by MRS2179 and a second unaffected component. The MRS2179-insensitive component was suppressed or abolished by inhibition of formation of nitric oxide by N(omega)-nitro-l-arginine methyl ester (l-NAME) in some, but not all, IJPs. An unidentified neurotransmitter, different from nitric oxide, mediated the second component in these cases. Nonpurinergic IJPs were a small third category (4%) of IJPs that were abolished by l-NAME and unaffected by MRS2179. Exogenous application of ATP evoked IJP-like hyperpolarizing responses, which were blocked by MRS2179. Application of apamin, which suppresses opening of small-conductance Ca(2+)-operated K(+) channels in the muscle, decreased the amplitude of the purinergic IJPs and the amplitude of IJP-like responses to ATP. The results support ATP as a neurotransmitter for IJPs in the intestine and are consistent with the hypothesis that the P2Y(1) purinergic receptor subtype mediates the action of ATP.     

Stretch-activated neuronal pathways to longitudinal and circular muscle in guinea pig distal colon

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells (<200 microm apart) at the oral region of the colon showed that excitatory junction potentials (EJPs) discharged synchronously in both muscle layers for periods of up to 6 h. Similarly, at the anal region of the colon, inhibitory junction potentials (IJPs) discharged synchronously in the two muscle layers. Quadruple recordings from LM and CM orally at the same time as from the LM and CM anally revealed that IJPs occurred synchronously in the LM and CM anally at the same time as EJPs in LM and CM located 20 mm orally. Oral EJPs and anal IJPs were linearly related in amplitude between the two muscle layers. Spatiotemporal maps generated from simultaneous video imaging of the movements of the colon, combined with intracellular recordings, revealed that some LM contractions orally could be correlated in time with IJPs in CM cells anally. N(omega)-nitro-L-arginine (L-NA; 100 microM) abolished the IJP in LM, whereas a prominent L-NA-resistant "fast" IJP was always observed in CM. In summary, in stretched preparations, synchronized EJPs in both LM and CM orally are generated by synchronized firing of many ascending interneurons, which simultaneously activate excitatory motor neurons to both muscle layers. Similarly, synchronized IJPs in both LM and CM anally are generated by synchronized firing of many descending interneurons, which simultaneously activate inhibitory motor neurons to both muscle layers. This synchronized motor activity ensures that both muscles around the entire circumference are excited orally at the same time as inhibited anally, thus producing net aboral propulsion.     

Inhibitory innervation of colonic smooth muscle cells and interstitial cells of Cajal

The effect of neural inhibition on the electrical activities of circular and longitudinal colonic smooth muscle was investigated. In addition, a comparative study was carried out between circular muscle preparations with and without the "submucosal" and "myenteric plexus" network of interstitial cells of Cajal (ICC) to study innervation of the "submucosal" ICC and to investigate whether or not the ICC network is an essential intermediary system for inhibitory innervation of smooth muscle cells. Electrical stimulation of intrinsic nerves in the presence of atropine caused inhibitory junction potentials (ijps) throughout the circular and longitudinal muscle layers. The ijp amplitude depended on the membrane potential and not on the location of the muscle cells with respect to the ICC network. Neurally mediated inhibition of the colon resulted in a reduction in amplitude and duration of slow wave type action potentials in circular and abolishment of spike-like action potentials in longitudinal smooth muscle, both resulting in a reduction of contractile activity. With respect to mediation by ICC, the study shows (i) "submucosal" ICC receive direct inhibitory innervation and (ii) circular smooth muscle cells can be directly innervated by inhibitory nerves without ICC as necessary intermediaries. The reversal potential of the ijp in colonic smooth muscle was observed to be approximately -76 mV, close to the estimated potassium equilibrium potential, suggesting that the nerve-mediated hyperpolarization is caused by increased potassium conductance.     

2-Aminoethoxydiphenyl borate blocks electrical coupling and inhibits voltage-gated K+ channels in guinea pig arteriole cells

2-Aminoethoxydiphenyl borate (2-APB) analogs are potentially better vascular gap junction blockers than others widely used, but they remain to be characterized. Using whole cell and intracellular recording techniques, we studied the actions of 2-APB and its potent analog diphenylborinic anhydride (DPBA) on vascular smooth muscle cells (VSMCs) and endothelial cells in situ of or dissociated from arteriolar segments of the cochlear spiral modiolar artery, brain artery, and mesenteric artery. We found that both 2-APB and DPBA reversibly suppressed the input conductance (G(input)) of in situ VSMCs (IC(50) ≈ 4-8 μM). Complete electrical isolation of the recorded VSMC was achieved at 100 μM. A similar gap junction blockade was observed in endothelial cell tubules of the spiral modiolar artery. Similar to the action of 18β-glycyrrhetinic acid (18β-GA), 2-APB and DPBA depolarized VSMCs. In dissociated VSMCs, 2-APB and DPBA inhibited the delayed rectifier K(+) current (I(K)) with an IC(50) of ～120 μM in the three vessels but with no significant effect on G(input) or the current-voltage relation between -140 and -40 mV. 2-APB inhibition of I(K) was more pronounced at potentials of ≤20 mV than at +40 mV and more marked on the fast component than on the slow component, which was mimicked by 4-aminopyridine but not by tetraethylammonium, nitrendipine, or charybdotoxin. In contrast, 18β-GA caused a linear inhibition of I(K) between 0 to +40 mV, which was similar to the action of tetraethylammonium or charybdotoxin. Finally, the 2-APB-induced inhibition of electrical coupling and I(K) was not affected by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C. We conclude that 2-APB analogs are a class of potent and reversible vascular gap junction blockers with a weak side effect of voltage-gated K(+) channel inhibition. They could be gap junction blockers superior to 18β-GA only when Ca(2+)-actived K(+) channel inhibition by the latter is a concern but inositol 1,4,5-trisphosphate receptor and voltage-gated K(+) channel inhibitions are not.     

Do gap junctions play a role in nerve transmissions as well as pacing in mouse intestine?

Varicosities of nitrergic and other nerves end on deep muscular plexus interstitial cells of Cajal or on CD34-positive, c-kit-negative fibroblast-like cells. Both cell types connect to outer circular muscle by gap junctions, which may transmit nerve messages to muscle. We tested the hypotheses that gap junctions transmit pacing messages from interstitial cells of Cajal of the myenteric plexus. Effects of inhibitors of gap junction conductance were studied on paced contractions and nerve transmissions in small segments of circular muscle of mouse intestine. Using electrical field stimulation parameters (50 V/cm, 5 pps, and 0.5 ms) which evoke near maximal responses to nitrergic, cholinergic, and apamin-sensitive nerve stimulation, we isolated inhibitory responses to nitrergic nerves, inhibitory responses to apamin-sensitive nerves and excitatory responses to cholinergic nerves. 18beta-Glycyrrhetinic acid (10, 30, and 100 microM), octanol (0.1, 0.3, and 1 mM) and gap peptides (300 microM of (40)Gap27, (43)Gap26, (37,43)Gap27) all failed to abolish neurotransmission. 18beta-Glycyrrhetinic acid inhibited frequencies of paced contractions, likely owing to inhibition of l-type Ca(2+) channels in smooth muscle, but octanol or gap peptides did not. 18beta-Glycyrrhetinic acid and octanol, but not gap peptides, reduced the amplitudes of spontaneous and nerve-induced contractions. These reductions paralleled reductions in contractions to exogenous carbachol. Additional experiments with gap peptides in both longitudinal and circular muscle segments after N(G)-nitro-l-arginine and TTX revealed no effects on pacing frequencies. We conclude that gap junction coupling may not be necessary for pacing or nerve transmission to the circular muscle of the mouse intestine.     

Disruption of smooth muscle gap junctions attenuates myogenic vasoconstriction of mesenteric resistance arteries

Communication between vascular smooth muscle (VSM) cells via low-resistance gap junctions may facilitate vascular function by synchronizing the contractile state of individual cells within the vessel wall. We hypothesized that inhibition of gap junctional communication would impair constrictor responses of mesenteric resistance arteries. Immunohistochemical experiments revealed positive staining for connexin 37 (Cx37) in both endothelium and smooth muscle of rat mesenteric arterioles, whereas connexin 43 (Cx43) immunoreactivity was not detected in the mesenteric vasculature. Administration of the gap junction inhibitory peptide Gap27, which targets Cx37 and Cx43, significantly diminished myogenic vasoconstriction (8.6 +/- 3.8% of passive diameter at 100 Torr) and changes in vessel wall intracellular [Ca2+] of mesenteric resistance arteries compared with vessels treated with either vehicle (physiological saline solution) (33.5 +/- 6.1%) or a control peptide (32.1 +/- 6.5%). Administration of 18alpha-glycyrrhetinic acid, structurally distinct from Gap27, also significantly attenuated myogenic constriction compared with its vehicle control (DMSO) (9.6 +/- 3.2% vs. 23.8 +/- 4.6%). In contrast, phenylephrine-induced vasoconstriction was not altered by gap junction blockers. Attenuated myogenic vasoconstriction resulting from inhibition of gap junctions persisted after disruption of the endothelium. In additional experiments, VSM cell membrane potential was recorded in mesenteric resistance arteries pressurized to 20 or 100 Torr. VSM membrane potential was depolarized at 100 Torr compared with 20 Torr. However, VSM cells in arteries treated with Gap27 were significantly hyperpolarized (-48.6 +/- 1.4 mV) at the higher pressure compared with vehicle (-41.4 +/- 1.5 mV) and Gap20-treated (-38.4 +/- 0.7 mV) vessels. Our findings suggest that inhibition of smooth muscle gap junctions attenuates pressure-induced VSM cell depolarization and myogenic vasoconstriction.     

Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor.

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.     

Gap junctional coupling between progenitor cells of regenerating retina in the adult newt

Gap junctional coupling between progenitor cells of regenerating retina in the adult newt was examined by a slice-patch technique. Retinal slices at the early regeneration stage comprised one to two layers of cells with mitotic activity, progenitor cells. These cells were initially voltage-clamped at a holding potential of -80 mV, near their resting potentials, and stepped to either hyperpolarizing or depolarizing test potentials under suppression of voltage-gated membrane currents. About half the cells showed passively flowing currents that reversed polarity around their resting potentials. The currents often exhibited a voltage- and time-dependent decline. As the difference between the test potential and resting potential increased, the time until the current decreased to the steady-state level became shorter and the amount of steady-state current decreased. Thus, the overall current profile was almost symmetrical about the current at the resting potential. Input resistance estimated from the initial peak of the currents was significantly smaller than that expected in isolated progenitor cells. In a high-K(+) solution, which decreased the resting potential to around 0 mV, the symmetrical current profile was also obtained, but only when the membrane potential was held at 0 mV before the voltage steps. These observations suggest that the current was driven and modulated by the junctional potential difference between the clamping cell and its neighbors. In addition, we examined effects of uncoupling agents on the currents. A gap junction channel blocker, halothane, suppressed the currents almost completely, indicating that the currents are predominantly gap junctional currents. Furthermore, injection of biocytin into the current-recorded cells revealed tracer coupling. These results demonstrate that progenitor cells of regenerating retina couple with each other via gap junctions, and suggest the presence of their cytoplasmic communication during early retinal regeneration.     

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon

The network of interstitial cells of Cajal associated with Auerbach’s (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach’s plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach’s plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities. Received: 21 July 1995 / Accepted: 22 April 1998     

Mechanism of active repolarization of inhibitory junction potential in murine colon

Enteric inhibitory responses in gastrointestinal (GI) smooth muscles involve membrane hyperpolarization that transiently reduce the excitability of GI muscles. We examined the possibility that an active repolarization mechanism participates in the restoration of resting membrane potential after fast inhibitory junction potentials (IJPs) in the murine colon. Previously, we showed these cells express a voltage-dependent nonselective cation conductance (NSCC) that might participate in active repolarization of IJPs. Colonic smooth muscle cells were impaled with micro-electrodes and voltage responses to nerve-evoked IJPs, and locally applied ATP were recorded. Ba2+ (500 muM), a blocker of the NSCC, slowed the rate of repolarization of IJPs. We also tested the effects of Ba2+, Ni2+, and mibefradil, all blockers of the NSCC, on responses to locally applied ATP. Spritzes of ATP caused transient hyperpolarization, and the durations of these responses were significantly increased by the blockers of the NSCC. We considered whether NSCC blockers might affect ATP metabolism and found that Ni2+ decreased ATP breakdown in colonic muscles. Mibefradil had no effect on ATP metabolism. Because both Ni2+ and mibefradil had similar effects on prolonging responses to ATP, it appears that restoration of resting membrane potential after ATP spritzes is not primarily due to ATP metabolism. Neurally released enteric inhibitory transmitter and locally applied ATP resulted in transient hyperpolarizations of murine colonic muscles. Recovery of membrane potential after these responses appears to involve an active repolarization mechanism due to activation of the voltage-dependent NSCC expressed by these cells.     

Synaptic transmission in simple motility reflex pathways excited by distension in guinea pig distal colon

We examined specific receptor/transmitter combinations used at functionally identified synapses in ascending and descending reflex pathways of guinea pig distal colon. Excitatory (EJPs) or inhibitory junction potentials (IJPs) were recorded intracellularly from nicardipine-paralyzed circular smooth muscle in either the oral or anal recording chamber of a three-chambered organ bath, respectively. Blockade of synaptic transmission in the central chamber with a 0.25 mM Ca2+/12 mM Mg2+ solution abolished EJPs evoked by distension applied either in the central or the far (anal) chamber. IJPs evoked by distension in the central or the far (oral) chamber were depressed to approximately 50% of control. Hexamethonium (nicotinic receptor antagonist, 200 microM) in the central chamber reduced IJPs evoked by far or central distension to 50%, whereas EJPs evoked by far distension were abolished and EJPs evoked by central distension were reduced to 70% of control. Hexamethonium in the recording chambers reduced both IJPs and EJPs evoked by central distension to approximately 50%. EJPs in the ascending pathway were unaffected by blockade of muscarinic receptors in the central chamber or blockade of neurokinin 3 tachykinin receptors in this or the recording chamber. In the descending pathway, blockade of P2 receptors in the same chambers had only a minor effect on distension-evoked IJPs. Thus some intrinsic sensory neurons of guinea pig colon have long descending projections (>30 mm), but ascending projections of <15 mm. In contrast to the ileum, transmission between ascending or descending interneurons and from sensory neurons to descending interneurons is predominantly via nicotinic receptors; but transmission to inhibitory or excitatory motoneurons and from sensory neurons to ascending interneurons involves nicotinic and other unidentified receptors.     

Role of Ca2+-activated Cl- channels and MLCK in slow IJP in opossum esophageal smooth muscle

The possible contribution of Ca2+-activated Cl- channel [I(Cl(Ca))] and myosin light-chain kinase (MLCK) to nonadrenergic, noncholinergic slow inhibitory junction potentials (sIJP) was studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body and guinea pig ileum perfused with Krebs solution containing atropine (3 microM), guanethidine (3 microM), and substance P (1 microM). In opossum esophageal circular smooth muscle, resting membrane potential (MP) was -51.9 +/- 0.7 mV (n = 89) with MP fluctuations of 1-3 mV. A single square-wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.3 +/- 0.2 mV, half-amplitude duration of 635 +/- 19 ms, and rebound depolarization amplitude of 2.4 +/- 0.1 mV (n = 89). 9-Anthroic acid (A-9-C), niflumic acid (NFA), wortmannin, and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) abolished MP fluctuations, sIJP, and rebound depolarization in a concentration-dependent manner. A-9-C and NFA but not wortmannin and ML-9 hyperpolarized MP. In guinea pig ileal circular smooth muscle, nerve stimulation elicited an IJP composed of both fast (fIJP) and slow (sIJP) components, followed by rebound depolarization. NFA (200 microM) abolished sIJP and rebound depolarization but left the fIJP intact. These data suggest that in the tissues studied, activation of I(Cl(Ca)), which requires MLCK, contributes to resting MP, and that closing of I(Cl(Ca)) is responsible for sIJP.     

Changes in the electrical activity of the rabbit proximal colon in vivo by stimulation of the vagus and splanchnic nerves]

1. Using extracellular electrodes placed on the serosa, we recorded the modifications of the electrical activity of the colonic muslce fibers caused by the stimulation of vagal and splanchnic nerve fibers. 2. Vagal stimulation produces two types of junction potentials: excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs). The IJPs are elicited by stimulation of vagal fibers which innervate intramural non-adrenergic inhibitory neurons. 3. The conduction velocity of the nerve impulse along the vagal pre-ganglionic fibers is 1.01 m/sec for excitatory fibers and 0.5. m/sec for inhibitory fibers. 4. Splanchnic fiber stimulation causes EJP disappearance, blocking transmission between preganglionic fibers and intramural excitatory neurons, and a decrease in IJP amplitude that most likely indicates a previous hyperpolarization of the smooth muscle. 5. IJP persistence during splanchnic stimulation proves that sympathetic inhibition does not modify the transmission of the vagal influx onto the non-adrenergic inhibitory neurons of the intramural plexuses. 6. Through a comparative study of proximal and distal colonic innervation, we are able to show that there is a similar organization of both regions, that is a double inhibitory innervation: an adrenergic one of a sympathetic origin, and a non adrenergic one of a parasympathetic origin.     

Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP?Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP?Rs in Ca(2+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(2+) sparks and Ca(2+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(2+) and constricted the arteries. The blockade of IP?Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(2+) sparks. Importantly, the IP?Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(2+) waves: Ca(2+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(2+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(2+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(2+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP?Rs in Ca(2+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.     

Generation of nonadrenergic inhibitory junction potentials in the smooth muscle of the guinea-pig gastrointestinal tract after replacing potassium ions with cesium ions

Nonadrenergic inhibitory junction potentials (IJPs), evoked by intramural nerve stimulation, were studied in the smooth muscle of the guinea-pig stomach, cecum, and colon, using a modified sucrose-gap technique. After incubating smooth muscle preparations for 4–9 h in potassium-free Krebs solution, IJPs were abolished, but reappeared when cesium ions (6 mM) were added to the Krebs solution. Under these conditions, in the majority of cases the amplitude of the IJP was half as small, and the latency and duration were significantly longer, than in normal conditions; also ATP, but not adenosine, caused hyperpolarization of the smooth muscle membrane. The amplitude of the IJP depended on the extracellular concentration of cesium. In all types of preparation, in cesium-containing Krebs solution, apamin usually abolished the IJP and responses to ATP. These results are consonant with the purinergic hypothesis of inhibitory neuromuscular transmission. The generation of the IJP in these potassium-free conditions depends on cesium ions, which pass through the small-conductance apamin-sensitive, calcium-dependent potassium channels.A. A. Bogomoletz Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 634–641, September–October, 1990.     

Structural differences in the enteric neural network in murine colon: impact on electrophysiology

The enteric neural network in the proximal murine colon shows a regularly occurring hypoganglionic region, which is here characterized by using anatomical and electrophysiological techniques. Staining with NADPH diaphorase, methylene blue, and cuprolinic blue in standard whole mounts and three-dimensional gut preparations of the murine proximal colon consistently revealed two hypoganglionic areas surrounded by a dense clustering of enteric neurons. This irregularity in the ganglionic plexus was found to be present in mice of three different genetic backgrounds, as well as in rats. The lack of myenteric ganglia in these regions was associated with an absence of the longitudinal muscle layer, as shown in cross sections. Histochemical identification of interstitial cells of Cajal in Kit(W-lacZ/+) transgenic mice showed Kit-positive cells oriented parallel to both muscle layers of the colon. Kit-positive cells oriented parallel to the longitudinal muscle layers were absent in the hypoganglionic area described. Electrical field stimulation elicited TTX-sensitive inhibitory junction potentials (IJPs), which showed region-specific characteristics. The initial partly apamin-sensitive hyperpolarization was present in all parts of the murine colon, whereas a second sustained NG-nitro-L-arginine-sensitive hyperpolarization was absent in the cecum and decreased from the proximal to the distal colon. Dissecting the hypoganglionic area from the surrounding tissue abolished the otherwise normal inhibitory neurotransmission to the circular muscle (1.6 +/- 1.4 and 2.6 +/- 1.7 mV for the fast and slow component of IJP amplitude in the hypoganglionic area vs. 16.5 +/- 1.9 and 23.7 +/- 2.7 mV for the fast and slow component of IJP amplitude in the neuron-rich area, respectively, P < 0.01, n = 6), whereas dissection of an area of identical size with an intact myenteric network showed normal inhibitory neurotransmission, indicating that the hypoganglionic area receives essential functional neural input from the neuron-rich surrounding tissue. In summary, in the murine and rat proximal colon, a constant and distinct hypoganglionic region is described with important concomitant changes in local electrophysiology.     

Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos.

Gap junctional proteins (connexins) form aqueous channels that enable direct cell-cell transfer of ions and small molecules. The distribution and conductance of gap junction channels in cardiac muscle determine the pattern and synchrony of cellular activation. However, the capacity for smooth muscle to restrict contractile events temporally and spatially suggests that cell-cell coupling or its regulation may be decidedly different in this tissue. We isolated a cDNA from vascular smooth muscle which encodes a connexin (Mr 43,187) structurally homologous to cardiac connexin43. Vascular smooth muscle connexin43 mRNA was expressed prominently in smooth muscle tissues, cultured vascular myocytes, and arterial endothelial cells. A model for functional expression of connexins was developed in two-cell B6D2 mouse embryos. Microinjection of in vitro transcribed vascular smooth muscle connexin43 mRNA was shown to be sufficient to induce intercellular coupling in previously uncoupled blastomeres. Through the construction of two deletion mutants of connexin43, we also show that the formation of cell-to-cell connections does not depend upon a predicted cytoplasmic region within 98 residues of the carboxyl terminus. Finally, the identification of connexin43 in smooth muscle and endothelial cells provides supporting evidence for the existence of heterocellular coupling between cells of the vascular intima.     

Synaptic inhibition of smooth muscles of the human colon: Effects of vitamin B6 and its derivatives

Spontaneous activity, which is manifested as slow depolarization waves and action potentials, is observed in most (81%) smooth muscles (SMs) of the circular layer of the human colon. Independently of the type of pathology, inhibitory junction potentials (IJPs) in SMs of various parts of the human colon are evoked by intramural stimulation; ranges of parameters of these potentials were comparable with those observed in muscle intestinal fragments isolated at a distance of several tens of centimeters from the zone of injury. In muscle strips (MSs) of such fragments, pyridoxal-5′-phosphate (PPh) applied in different concentrations caused suppression of IJPs: in the concentration of 1·10⁻⁸ to 1·10⁻⁴ M it decreased the amplitude, and in the concentrations of 1·10⁻⁵ to 1·10⁻⁴ M and 1·10⁻⁴ M, respectively, it decreased rates of the half-amplitude rise and decay of these potentials. Pyridoxal (1·10⁻⁴ M) and 4-pyridoxolic acid (1·10⁻⁴ M) also caused a drop in the amplitude of IJPs; however, these agents influenced this parameter to a lesser extent, as compared with the effect of 1·10⁻⁴ M PPh. Pyridoxine (1·10⁻⁴ M) and pyridoxamine (1·10⁻⁴ M) evoked no significant changes in the parameters of IJPs in MSs of the human colon. Our data allow us to hypothesize that the suppressing effect of PPh on IJPs is determined by the presence of a purinergic component present in non-adrenergic inhibition of SMs of the human colon. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 269–279, July–August, 2006.