The gaseous molecule hydrogen sulfide (H₂S) has been proposed as an endogenous signal molecule and neuromodulator in mammals. Using a newly developed method, we report here for the first time the ...ability of intact and living brain and colonic tissue in the mouse to generate and release H₂S. This production occurs through the activity of two enzymes, cystathionine-γ-lyase and cystathionine-β-synthase. The quantitative expression of messenger RNA and protein localization for both enzymes are described in the liver, brain, and colon. Expression levels of the enzymes vary between tissues and are differentially distributed. The observation that, tissues that respond to exogenously applied H₂S can endogenously generate the gas, strongly supports its role as an endogenous signal molecule.
Background & Aims Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced ...damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic NOD) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay. Methods Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction. Results Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206+ M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206+ /HO1+ M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206+ /HO1+ M2 macrophages. Conclusions CD206+ M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.
Sodium channels are key regulators of neuronal and muscle excitability. However, sodium channels have not been definitively identified in gastrointestinal smooth muscle. The aim of the present study ...was to determine if a Na+ current is present in human jejunal circular smooth muscle cells.
Currents were recorded from freshly dissociated cells using patch-clamp techniques. Complementary DNA (cDNA) libraries constructed from the dissociated cells were screened to determine if a message for α subunits of Na+ channels was expressed. Smooth muscle cells were also collected using laser-capture microdissection and screened.
A tetrodotoxin-insensitive Na+ channel was present in 80% of cells patch-clamped. Initial activation was at −65 mV with peak inward current at −30 mV. Steady-state inactivation and activation curves revealed a window current between −75 and −60 mV. The Na+ current was blocked by lidocaine and internal and external QX314. A cDNA highly homologous to SCN5A. the α subunit of the cardiac Na+ channel, was present in the cDNA libraries constructed from dissociated cells and from smooth muscle cells collected using laser-capture microdissection.
Human jejunal circular smooth muscle cells express a tetrodotoxin-insensitive Na+ channel, probably SCN5A. Whether SCN5A plays a role in the pathophysiology of human gut dysmotilities remains to be determined.
The paraventricular nucleus (PVN) of the hypothalamus plays an important role in regulating gut motility. To date, there have been no intracellular electrophysiological studies of dog PVN neurons in ...vitro. The aims of this study were to: (1) adapt brain slice methods developed for studies of rodent CNS tissue to canine CNS tissue; and (2) study the electrophysiology and morphology of single neurons of the dog paraventricular nucleus (PVN). Coronal hypothalamic slice preparations (400 μm thick) of dog brain were used. Three groups of PVN neurons were classified based on their firing pattern. Continuous firing neurons (
n=32) exhibited continuous ongoing action potentials (APs). Burst firing neurons generated bursts of APs (
n=19). Intermittent firing neurons had only a few spontaneous APs. In contrast to continuous firing neurons, 14 of 19 burst firing neurons and 3 of 7 intermittent firing neurons responded to depolarizing current with a Ca
2+-dependent low-threshold potential. Twenty-one PVN neurons studied electrophysiologically were filled with biocytin. Continuous firing neurons (
n=12) had oval-shaped soma with two or three sparsely branched dendrites. Branched axons were found in two continuous firing neurons, in which one branch appeared to terminate locally. Burst firing neurons (
n=8) generally had triangular soma with 2 to 5 branched dendrites. In summary, the brain slice technique was used to study the morphology and electrophysiology of single neurons of the dog brain. Electrophysiological and morphological properties of the three neuron groups were identified and discussed.
Electrical activity and synaptic responses were recorded intracellularly in 415 neurons of the mouse superior mesenteric ganglion (SMG) attached to a segment of distal colon in vitro. Eighty-seven ...percent of neurons tested received ongoing nicotinic cholinergic fast excitatory postsynaptic potentials (fEPSPs). Colonic distension caused an initial transient followed by a sustained, slowly adapting increase in fEPSP activity. Application of hexamethonium only to the colon reduced, but did not completely abolish, distension-evoked responses, suggesting direct projection of some distension-sensitive fibers. Ongoing fEPSPs were abolished when nerve trunks connecting the SMG to the colon were transected or blocked with tetrodotoxin applied to the colon. Intracellular labeling with horseradish peroxidase or lucifer yellow revealed that about 90% of neurons receiving colonic synaptic input had a caudally projecting axon; about 60% that did not receive colonic input had a rostrally projecting axon. The latter neurons were found only in the cephalad ganglion region. These results show that mouse SMG neurons receive colonic mechanosensory afferent synaptic input and thus may participate in sympathetic intestinal reflexes.
Cavernous smooth muscle cells have a key role in the control of penile erection and detumescence. In this study the types of smooth muscle cells and currents present in isolated rabbit corpus ...cavernosum myocytes were characterized.
Immunohistochemical methods were used to identify cavernous smooth muscle cells. Currents were recorded from freshly dissociated myocytes using the whole cell and amphotericin perforated patch clamp techniques.
Cavernous myocytes were identified by α-smooth muscle actin and smooth muscle myosin immunoreactivity. Based on electrical properties at least 2 types of myocytes were present. Type I cells showed more depolarized membrane potentials, lower capacitance, higher input resistance and increased current densities at positive potentials than type II cells. In types I and II cells at voltages positive to 30 mV, maxi K+ channel (Ca
2+ activated large conductance K+ channel or BK) blockade with iberiotoxin or charybdotoxin reduced outward currents by approximately 40% to 80% at 80 mV. Maxi K+ channel blocking did not affect cell membrane potential. Type II cells showed delayed rectifier K+ channel-type outward currents that were not detected in type I cells. Delayed rectifier K+ channel-type currents were resistant to iberiotoxin or charybdotoxin, activated at approximately −50 to −40 mV. and inactivated weakly.
The data suggest that cavernous smooth muscle cells are heterogeneous with at least 2 subtypes identified based on membrane potential, capacitance, input resistance, current density and delayed rectifier K+ channel expression. The activation threshold suggests that delayed rectifier K+ channels are open at the resting membrane potential and, therefore, contribute to control and regulation of the cavernous myocyte excitability.
Intracellular recordings were made from cells located in the longitudinal, inner and outer circular muscle layers of the dog,
cat, rabbit, opossum and human small intestine. In whole-thickness ...preparations in all five species, longitudinal muscle cells
generated slow waves and spikes. However, in isolated longitudinal muscle preparations, all cells tested were electrically
silent. In whole-thickness and in isolated preparations, cells in the inner circular muscle layer generated spontaneous spikes
superimposed on slow potentials. However, the occurrence of spikes and slow potentials was more regular in whole-thickness
preparations. In whole-thickness preparations, cells in the outer circular muscle layer generated slow waves which were coupled
with phasic contractions. However, in isolated outer circular muscle preparations, all cells tested were electrically silent
and spontaneous phasic contractions were absent. In whole-thickness preparations, non-neural cells located on the serosal
side of the outer circular muscle layer generated slow waves. The data suggest that spontaneous slow waves of the small intestine
of the dog, cat, rabbit, opossum and human are generated in non-neural cells located between the longitudinal and outer circular
muscle layer and by non-neural cells located between the outer and inner circular muscle layers.
The aim of this study was to determine whether leptin modulates neuronal activity in intrapancreatic ganglion neurons. Intracellular recordings were made in dog pancreatic neurons. Recombinant mouse ...leptin (313 nM) was added by superfusion. When leptin was present, fast EPSPs which were subthreshold in normal Krebs solution reached threshold for firing action potentials. However, leptin had no significant (
P>0.05,
n=18) effect on either the resting membrane potential or on membrane input resistance. To determine whether leptin increased the postsynaptic sensitivity to acetylcholine, the response was tested by pressure ejection of acetylcholine. Acetylcholine evoked a 9.4±2.2 mV (mean±SEM,
n=5) depolarization in normal Krebs solution. In the presence of leptin, the response was not significantly different (9.6±2.4 mV,
P>0.05). The results suggest that leptin modulates fast synaptic transmission in pancreatic ganglion neurons by acting on presynaptic nerve terminals.
Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from ...human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the alpha(1C) L-type calcium channel subunit (Ca(V)1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a beta(2) calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac alpha(1C) splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the alpha(1C) COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming alpha(1C)-subunit.
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