Katsuhide Nishi

C-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a netword similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.

Blockade of Kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype

BACKGROUND & AIMS Interstitial cells of Cajal (ICC) serve as pacemaker cells and mediators of neurotransmission from the enteric nervous system to gastrointestinal muscles. ICC develop from mesenchymal cells that express c-Kit, and signaling via Kit receptors is necessary for normal development of ICC. We studied the fate of functionally developed ICC after blockade of Kit receptors to determine whether ICC undergo cell death or whether the phenotype of the cells is modified. The fate of undeveloped ICC was also investigated. METHODS Neutralizing, anti-Kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. ICC in the small intestine were examined by immunohistochemistry and electron microscopy. Occurrence of apoptosis was also assayed. RESULTS When Kit receptors were blocked, ICC nearly disappeared from the small intestine. Apoptosis was not detected in regions where ICC are normally distributed. Remaining Kit-immunopositive cells in the pacemaker region of the small intestine developed ultrastructural features similar to smooth muscle cells, including prominent filament bundles and expression of the muscle-specific intermediate filament protein, desmin, and smooth muscle myosin. ICC of the deep muscular plexus normally develop after birth in the mouse. Precursors of these cells remained in an undifferentiated state when Kit was blocked. CONCLUSIONS These data, along with previous studies showing that ICC in the pacemaker region of the small intestine and longitudinal muscle cells develop from the same Kit-immunopositive precursor cells, suggest inherent plasticity between the ICC and smooth muscle cells that is regulated by Kit-dependent cell signaling.

Rhythmic Cl− current and physiological roles of the intestinal c-kit-positive cells

Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit+) cells when studied immuno-histochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c -kit+ cells, we studied the excitability of intestinal c -kit+ cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltageclamp at −40 mV, the majority of c -kif+cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263±24 pA and 2.30±0.25 cycles/min (mean±SEM), respectively. Intracellular perfusion of the c -kit+ cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca2+-free external solution or low holding voltage (<-60 mV) prevented the rhythmic current. The reversal potential of the rhythmic current was close to the equilibrium potential for Cl−(ECl) Moreover the rhythmic current was depressed by a Cl− channel blocker, 4-acetoamido-4-isothiocyanat-ostilbene-2,2′-disulphonic acid (SITS). The smooth muscle cells freshly dissociated from the same intestinal specimen revealed a Ca2+-activated K+current, as has been described in a variety of smooth muscle cells. Cultured smooth muscle cells from the ileum preparation lacked neither the Ca2+-activated K+nor rhythmic Cl− currents. Smooth muscle cells freshly dissociated from the same ileum preparation and those in culture showed no immunoreactivity with the labelled ACK2, which was consistent with our previous in situ study. Results provided direct evidence that the intestinal c -kit+ cells, but not the smooth muscle cells, possess a rhythmic Cl− current oscillation, suggesting their participation in pacemaker activity for the peristaltic gut movement.

Activation of afferent cardiac sympathetic nerve fibers of the cat by pain producing substances and by noxious heat

SummaryAfferent discharges of 64 single units were recorded from the left cardiac sympathetic nerve of anesthetized cats. Mechano-sensitive terminals of the afferent fibers were localized in the extrapulmonary part of the pulmonary artery, left atrium, left ventricle and left pericardium, as determined by direct mechanical probing of the heart after death of the animals. Conduction velocity of the fibers ranged from 2.5 to 14.6 m/s. Excitation of these Aδ-fibers with mechanically excitable endings was produced by intravenous injections of acetylcholine, 5-hydroxytryptamine, bradykinin, histamine and veratridine, and/or by topical application of these agents to the receptor region. Noxious heat to the mechanically excitable field in the wall of the pulmonary artery and the left ventricle also activated their afferent fibers. These observations provide evidence for a certain number of afferent units in the cardiac sympathetic nerve with polymodal sensitivity. These afferent fibers can provide the spinal cord with information not only on mechanical changes in cardiac events, but also changes in the chemical environment of the cardiac nerve ending, possibly produced by myocardial ischemia.

Actions of verapamil, diltiazem and other divalent cations on the calcium-current of Helix neurones

1 Effects of organic Ca2+‐antagonists, verapamil and diltiazem, and cations, Ni2+, Mn2+, Co2+ and La3+ on Ca2+ current (ICa) separated from other ionic currents in a Helix neurone were studied. A suction pipette technique which allows internal perfusion of the cell body and voltage clamp was used 2 Verapamil and diltiazem (10−6‐10−4m) increased the threshold, and decreased both the amplitude and rate of rise of the soma Ca2+‐spike. Both agents inhibited ICa over the entire range of the current‐voltage (I‐V) relationship dose‐dependently, without shifting the threshold of the I‐V relationship. Increases in external Ca2+ overcame the inhibitory action of the agents 3 Divalent cations, Ni2+, Mn2+, Co2+ and the trivalent cation, La3+ inhibited ICa dose‐dependently, but induced shifts of the I‐V relationship to more positive voltages. The order of potency of inhibition of ICa among these cations was as follows; Ni2+ > La3+ > Mn2+ > Co2+ 4 Double reciprocal plots for peak ICa versus external Ca2+ concentrations in the presence or absence of both organic and inorganic Ca2+‐antagonists intersect at the ordinate. Results indicate that both organic and inorganic Ca2+‐antagonists compete for Ca2+ at the common binding site for Ca2+ 5 Internal application of the organic Ca2+‐antagonists (10−4m) inhibited ICa in a time‐dependent manner to about 40–60% of the control. Ni2+, when applied internally, also depressed ICa 6 The results provide evidence that organic Ca2+‐antagonists occupy the binding site for Ca2+ in a competitive manner at the surface of the soma membrane of the Helix neurone, while divalent and trivalent cations, in addition to inhibiting ICa in a similar manner to the organic Ca2+‐antagonists, change the surface charge of the soma membrane.

THE ACTION OF 5‐HYDROXYTRYPTAMINE ON CHEMORECEPTOR DISCHARGES OF THE CATS CAROTID BODY

1 Chemoreceptor discharges were recorded in vivo from fine filaments of the carotid sinus nerve containing a single or several active units; their frequency was used as an index of receptor activity. The effects of 5‐hydroxytryptamine (5‐HT) on chemoreceptors were studied in 26 adult cats. At times, sinus baroreceptor discharges were recorded from the carotid nerve and the effect of 5‐HT on the discharges was examined. 2 Intra‐carotid injections of 5‐HT (2–20 μg) induced a sharp and brief increase in chemoreceptor discharges, followed by depression or block which lasted for several seconds. Repeated injections at short intervals, and a small dose after a large dose of 5‐HT resulted in depressed or blocked response to 5‐HT. 3 5‐HT in high doses (10–20 μg, La.) slightly depressed the chemoreceptor discharges induced by either acetylcholine (ACh) or NaCN, when these substances were applied within 20 s after 5‐HT. 5‐HT (5–20 μg, i.a.) applied during asphyxia induced a further increase in chemoreceptor discharges, soon followed by block of the discharges lasting for several seconds. 4 Atropine or hexamethonium in high doses did not change the chemoreceptor response to 5‐HT, while that to ACh was markedly depressed. 5 (+)‐Lysergic diethylamide (LSD), methysergide or gramine did not alter the response to 5‐HT, while LSD in low doses produced a marked increase in chemoreceptor discharges. 6 Acute and chronic treatment with reserpine (5–10 mg/kg, i.v.) of the animals did not change the sensitivity and the reactivity of the chemoreceptor to ACh and NaCN, while the chemoreceptor response to 5‐HT was augmented, indicating an increase in the sensitivity of chemoreceptors to 5‐HT. 7 5‐HT in small doses (2–10 μg, i.a.) induced a marked increase in sinus baroreceptor discharges; subsequently discharges were depressed or blocked for several seconds. 8 The results are discussed in relation to possible mechanism of action of 5‐HT on the chemoreceptors. It is concluded that the exogenous 5‐HT probably acts directly on the chemosensory nerve endings and depolarizes them, but 5‐HT contained in the carotid body does not play a significant role in the generation of chemoreceptor discharges.

FURTHER ANALYSIS OF INHIBITORY EFFECTS OF PROPRANOLOL AND LOCAL ANAESTHETICS ON THE CALCIUM CURRENT IN Helix NEURONES

1 The effects of propranolol and local anaesthetics on Ca2+ current (ICa), individually separated from other ionic currents, in Helix neurones were studied under voltage clamp, using a suction pipette technique. 2 Increases in external Ca2+ concentrations overcame the inhibitory action of propranolol on lea‐Double reciprocal plots for peak ICa versus external Ca2+ concentrations in the presence or absence of propranolol did not intersect at the ordinate. 3 Internal application of propranolol (10−4m) inhibited ICa to about 40–60% of the control in a time‐dependent manner. 4 Lignocaine and procaine at concentrations of 10−3–10−2m inhibited ICa without shifting the threshold in the I‐V relationships. Internal application of lignocaine (10−3–10−2 m) also inhibited ICa: the ratio of depression of the ICa was almost equivalent to that of the agent applied externally. 5 The results provide evidence that propranolol inhibits ICa in a noncompetitive manner with Ca2+ at the cell membrane, and suggest that the agents may occupy the receptor site in the Ca2+ ‐channel somewhere between the outer surface and inner phase of the membrane.

An Amiloride-Sensitive and Voltage-Dependent Na+ Channel in an HLA-DR-Restricted Human T Cell Clone

We investigated changes in voltage-gated Na+ currents and effects of extracellular Na+ on proliferation in HLA-DR-restricted human CD4+ αβ T cells after stimulation with a non-self antigenic peptide, M12p54–68. In the absence of antigenic peptide, neither single (n = 80) nor APC-contacted (n = 71) T cells showed voltage-gated inward currents recording with whole-cell patch-clamp techniques, even with Ca2+ and Na+ ions present in the perfusion solution. However, with the same recording conditions, 31% (26 of 84) of APC-contacted T cells stimulated with the antigenic peptide showed voltage-dependent inward currents that were elicited from −60 mV. The inward currents were not inhibited in extracellular Ca2+-free conditions or in the presence of 1 mM NiCl2. However, they were completely inhibited in extracellular Na+-free conditions, which were made by replacing Na+ with iso-osmotic N-methyl-d-glucamine or choline. The Na+ currents were insensitive to tetrodotoxin, a classical blocker of Na+ channels, but were dose-dependently inhibited by amiloride, a potassium-sparing pyrazine diuretic. Furthermore, the Ag-specific proliferative response of T cells was completely inhibited in Na+-free Tyrode’s solution and was suppressed by amiloride in a dose-dependent manner. Our findings suggest that activation of amiloride-sensitive and voltage-gated Na+ channels would be an important step to allow an adequate influx of Na+ and maintain a sustained high Ca2+ level during T cell activation.

Heparin adheres to the damaged arterial wall and inhibits its thrombogenicity.

Heparin binds to thrombogenic extracellular matrices as well as to smooth muscle cells of the vascular wall in vitro. The inhibitory effects of heparin on thrombogenicity of the damaged arterial wall were examined in vivo using small mesenteric arteries of rats and a video recording system attached to a microscope. To induce thrombosis, we damaged the vessel wall over a short segment by compression and exposed the media to the blood stream. A platelet-rich thrombus enlarged gradually at the damaged site, occluded the vascular lumen for a short period, and then flowed away. Compression damage induced such thrombus formation several times. Heparin (500 units/ml) was given in three different ways: intravenous and intra-arterial administration (both 300 units/kg) and intraluminal application under stopped-flow conditions (less than 0.01 ml) for 1-2 minutes with subsequent draining out. Intravenous heparin significantly decreased both the total duration and the number of thrombotic occlusions, whereas intra-arterial heparin abolished thrombotic occlusion. Both routes of heparin administration similarly prolonged the blood coagulation time. Intraluminal application of heparin significantly inhibited subsequent thrombus formation after restoring the flow without changes in the blood coagulation time. After an intra-arterial administration or intraluminal application of fluorescein isothiocyanate-bound heparin, strong fluorescence was observed only at the damaged vascular segment. A heparin fraction with low affinity to antithrombin III or chondroitin sulfate A did not inhibit thrombosis. To clarify anticoagulant activity of vascular wall-bound heparin, damaged carotid arterial segments of rats were incubated (inside out) in platelet-poor plasma with thrombin, and fibrin clot formation around the segments with or without heparin binding was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular chloride activity increases in guinea pig ventricular muscle during simulated ischemia

We investigated the effects of simulated ischemia on intracellular Cl- activity ([Cl-]i) in isolated guinea pig ventricular papillary muscles using ion-selective microelectrode techniques. Simulated ischemia in ventricular muscles was produced by stopping the flow of superfusion and immersing preparations in mineral oil as previously described [B. Vanheel, L. Leybaert, A. De Hemptinne, and I. Leusen. Am. J. Physiol. 257 (Cell Physiol. 26): C365-C379, 1989; Z. F. Lai, J. Liu, and K. Nishi. Jpn. J. Pharmacol. 72: 161-174, 1996]. When preparations were exposed to paraffin oil for 15 min, [Cl-]i markedly increased and the peak magnitude of [Cl-]i reached 55.3 +/- 2.5 mM from 18.7 +/- 3.5 mM, whereas membrane potentials (Vm) depolarized from -82.5 +/- 1.1 to -54.7 +/- 2.4 mV (n = 6 muscles from 6 animals). SITS (0.5 mM), a known blocker of the Cl-/HCO-3 exchanger, suppressed the ischemia-induced depolarization of Vm and delayed the onset of the ischemia-induced increase in [Cl-]i but did not suppress the magnitude of the increase of [Cl-]i. Under Cl--free conditions created by replacing Cl- with equimolar gluconate, the increase in [Cl-]i during ischemia was transient and suppressed by >60% compared with that in normal-Cl- conditions (peak value was 20. 3 +/- 1.7 mM, n = 6 muscles from 6 animals). The present results provide direct evidence that [Cl-]i in ventricular muscle increases in ischemic conditions in quiescent guinea pig ventricular muscle, suggesting that activation of the Cl-/HCO-3 exchanger by ischemia would partially contribute to the elevation of [Cl-]i during the initial stage of ischemia.We investigated the effects of simulated ischemia on intracellular Cl- activity ([Cl-]i) in isolated guinea pig ventricular papillary muscles using ion-selective microelectrode techniques. Simulated ischemia in ventricular muscles was produced by stopping the flow of superfusion and immersing preparations in mineral oil as previously described [B. Vanheel, L. Leybaert, A. De Hemptinne, and I. Leusen. Am. J. Physiol. 257 ( Cell Physiol. 26): C365-C379, 1989; Z. F. Lai, J. Liu, and K. Nishi. Jpn. J. Pharmacol. 72: 161-174, 1996]. When preparations were exposed to paraffin oil for 15 min, [Cl-]imarkedly increased and the peak magnitude of [Cl-]ireached 55.3 ± 2.5 mM from 18.7 ± 3.5 mM, whereas membrane potentials ( V m) depolarized from -82.5 ± 1.1 to -54.7 ± 2.4 mV ( n = 6 muscles from 6 animals). SITS (0.5 mM), a known blocker of the Cl-/[Formula: see text]exchanger, suppressed the ischemia-induced depolarization of V m and delayed the onset of the ischemia-induced increase in [Cl-]ibut did not suppress the magnitude of the increase of [Cl-]i. Under Cl--free conditions created by replacing Cl-with equimolar gluconate, the increase in [Cl-]iduring ischemia was transient and suppressed by >60% compared with that in normal-Cl- conditions (peak value was 20.3 ± 1.7 mM, n = 6 muscles from 6 animals). The present results provide direct evidence that [Cl-]iin ventricular muscle increases in ischemic conditions in quiescent guinea pig ventricular muscle, suggesting that activation of the Cl-/[Formula: see text]exchanger by ischemia would partially contribute to the elevation of [Cl-]iduring the initial stage of ischemia.