Relaxation induced by N-terminal fragments of chromogranin A in mouse gastric preparations

A definitive role for chromogranin A (CGA)-derived fragments in the control of the gastrointestinal smooth muscle contractility has not been yet established. The purpose of the present study was to evaluate, in vitro, the effects of the recombinant vasostatin 1-78 (VS-1), CGA 7-57 and CGA 47-66 on the mouse gastric mechanical activity, recording the changes of intraluminal pressure. VS-1, CGA 7-57 and CGA 47-66 produced concentration-dependent relaxations. Mouse anti-vasostatin-1 monoclonal antibody 5A8, recognising the region 53-57, abolished the relaxation induced by VS-1, indicating the specificity of the effect. The relaxation was significantly reduced by tetrodotoxin (TTX), blocker of neuronal voltage-dependent Na(+) channels, l-NAME, inhibitor of nitric oxide (NO) synthase, or apamin, blocker of small conductance Ca(2+)-dependent K(+) channels. The joint application of TTX and l-NAME did not show any additive effects, whereas TTX plus apamin abolished the VS-1 response. The results suggest that the N-terminal CGA-derived peptides are able to relax mouse gastric muscle and, therefore, they point out an inhibitory role of vasostatin I in the gastrointestinal tract. The relaxation is mediated in part by neural mechanisms through NO production and in part by non-neural mechanisms involving the opening of small conductance Ca(2+)-dependent K(+) channels.     

Contribution of NK(2) tachykinin receptors to propulsion in the rabbit distal colon

The role of the tachykinin neurokinin (NK)(2) receptors on rabbit distal colon propulsion was investigated by using two selective NK(2)-receptor antagonists, MEN-10627 and SR-48968. Experiments on colonic circular muscle strips showed that contractile responses to [beta-Ala(8)]NKA-(4-10) (1 nM-1 microM), a selective NK(2)-receptor agonist, were competitively antagonized by MEN-10627 (1-100 nM), whereas SR-48968 (0.1-10 nM) caused an insurmountable antagonism, thus confirming the difference in the mode of action of the two compounds. Colonic propulsion was elicited by distending a mobile rubber balloon with 0.3 ml (submaximal stimulus) or 1.0 ml (maximal stimulus) of water. The velocity of anal displacement of the balloon (mm/s) was considered the main propulsion parameter. At low concentrations (1.0-100 nM and 0.1-10 nM, respectively), MEN-10627 and SR-48968 facilitated the velocity of propulsion, whereas at high concentrations (100 nM and 1 microM, respectively) they decelerated propulsion. The excitatory and inhibitory effects of both antagonists were observed only with submaximal stimulus. We focused on the hypothesis that the facilitatory effect on propulsion may result from blockade of neuronal NK(2) receptors and the inhibitory effect from suppression of the excitatory transmission mediated by NK(2) receptors on smooth muscle cells. In the presence of N(G)-nitro-L-arginine (300 microM), a nitric oxide synthase inhibitor, MEN-10627, at a concentration (10 nM) that was found to accelerate propulsion in control experiments inhibited the velocity of propulsion. In the presence of threshold (1-10 nM) or full (1 microM) concentration of atropine, which inhibited to a great extent the velocity of propulsion, the inhibitory effect of MEN-10627 (1 microM) was markedly increased. In conclusion, in the rabbit distal colon NK(2) receptors may decelerate propulsion by activating a nitric oxide-dependent neuronal mechanism and may accelerate it by a postjunctional synergistic interaction with cholinergic muscarinic receptors.     

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.     

Muscarinic regulation of neonatal rat bladder spontaneous contractions

In vitro preparations of whole urinary bladders of neonatal rats exhibit prominent myogenic spontaneous contractions, the amplitude and frequency of which can be increased by muscarinic agonists. The muscarinic receptor subtype responsible for this facilitation was examined in the present experiments. Basal spontaneous contractions in bladders from 1- to 2-wk-old Sprague-Dawley rats were not affected by M2 or M3 receptor antagonists. However, administration of 0.5 microM physostigmine, an anticholinesterase agent that increases the levels of endogenous acetylcholine, or 50-100 nM carbachol, a cholinergic agonist at low concentrations, which did not cause tonic contractions, significantly augmented the frequency and amplitude of spontaneous contractions. Blockade of M2 receptors with 0.1 microM AF-DX 116 or 1 microM methoctramine or blockade of M3 receptors with 50 nM 4-diphenylacetoxy-N-methylpiperidine methiodide or 0.1 microM 4-diphenylacetoxy-N-(2-chloroethyl)piperidine hydrochloride (4-DAMP mustard) reversed the physostigmine and carbachol responses. M2 and M3 receptor blockade did not alter the facilitation of spontaneous contractions induced by 10 nM BAY K 8644, an L-type Ca2+ channel opener, or 0.1 microM iberiotoxin, a large-conductance Ca2+-activated K+ channel blocker. NS-1619 (30 microM), a large-conductance Ca2+-activated K+ channel opener, decreased carbachol-augmented spontaneous contractions. These results suggest that spontaneous contractions in the neonatal rat bladder are enhanced by activation of M2 and M3 receptors by endogenous acetylcholine released in the presence of an anticholinesterase agent or a cholinergic receptor agonist.     

Arecoline excites the colonic smooth muscle motility via M3 receptor in rabbits

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.     

Effects of cholecystokinin octapeptide and somatostatin on the motility and release of [3H]acetylcholine in canine colon

1. The longitudinal and circular muscle layers of canine colon showed a different pattern of mechanical activity: regular rhythmic phasic contractions in the circular strips and irregular rhythmic prolonged contractions in the longitudinal strips.2. The spontaneous motility of both layers was suppressed by atropine (1 μM) or hexamethonium (1 μM), suggesting the involvement of ACh.3. Somatostatin (1 nM–1μM) decreased, while CCK8 (1–10 nM) increased the spontaneous and electrically-induced contractions of the colonic muscles, the circular layer being more sensitive as compared to the longitudinal layer.4. CCK8 enhanced both resting and electrically-induced [³H]ACh release, while SOM inhibited the electrically-stimulated [³H]ACh release.     

Involvement of K(+)-channel opening in endothelin-1 induced suppression of spontaneous contractions in the guinea pig taenia coli.

Effects of porcine-human endothelin-1 on mechanical as well as electrical activities and on intracellular free Ca2+ levels in the guinea pig taenia coli were compared with those of nifedipine, a voltage-dependent Ca2+ channel blocker. Endothelin-1 (0.1-100 nM) caused a concentration-dependent suppression of spontaneous contractions but did not significantly affect the sustained contraction evoked by 40 mM KCl. However, nifedipine (0.1-100 nM) inhibited both types of contractions in a concentration-dependent manner. In electrophysiological studies, endothelin-1 (30 nM) or nifedipine (30 nM) eliminated spontaneous spike discharges. Endothelin-1 produced hyperpolarization, while nifedipine did not change the resting membrane potential. The endothelin-1 induced suppression of spontaneous contractions was dose-dependently antagonized by apamin (0.01-10 nM), an inhibitor of a small conductance Ca(2+)-dependent K+ channel, and D-tubocurarine (10-100 microM), an inhibitor of Ca(2+)-dependent K+ channel, but was unaffected by 4-aminopyridine (0.01-1 mM), an inhibitor of a voltage-dependent K+ channel. In the study with fura 2 excited at 340 nm, endothelin-1 abolished, from the tissue, the fluorescence signals that were coupled with spontaneous contraction. It is suggested that the inhibitory action of endothelin-1 on spontaneous contraction may be caused by hyperpolarization of the membrane that reduces the spontaneous generation of spike discharge coupled normally to an increase in the intracellular free Ca2+ levels in the guinea pig taenia coli. The hyperpolarization may be caused by activating apamin-sensitive Ca(2+)-dependent K+ channels.     

Time course of neural and contractile disturbances in a rat model of colitis induced by Trichinella spiralis

Colitis induced by Trichinella spiralis in rat induces alterations in the spontaneous motor pattern displayed by circular colonic muscle [Auli, M., Fernandez, E., 2005. Characterization of functional and morphological changes in a rat model of colitis induced by T. spiralis. Digestive Diseases and Sciences 50(8), 1432-1443]. We examined the temporal relationship between the severity of inflammation and the altered contractility of the underlying circular muscle as well as the role of NANC inhibitory pathways in the disruption of the motility pattern. Colitis was induced by intrarectal administration of T. spiralis larvae. Responses to acetylcholine (ACh) and increased extracellular potassium as well as the effect of tetrodotoxin (TTX, 1 microM), N-nitro-l-arginine (L-NOARG, 1 mM) and apamin (1 microM) were determined in vitro in the organ bath with circular muscle strips from sham-infected and infected rats at days 2-30 postinfection (PI). Microelectrode recordings were performed to study the putative changes in electrical activity of colonic smooth muscle cells. Responses to ACh and KCl were decreased at all days PI compared to sham. Intracellular calcium depletion had a greater inhibitory effect in inflamed tissue (6-14 PI). The effect of TTX, L-NOARG and apamin on the spontaneous contractions was found to be altered in all infected rats, i.e. their effects were transient and milder. Inflamed tissue showed lower resting membrane potential and a decreased duration of inhibitory junction potentials induced by electrical stimulation. These data suggest that the decreased contractility of colonic circular smooth muscle induced by the intrarectal T. spiralis infection results from the impairment of the excitation-contraction coupling, from a persistent hyperpolarization of smooth muscle cells and from impaired NANC inhibitory neurotransmission.     

Contractile effects of 16-methyl analogues of PGE2 on the circular and longitudinal muscles of the guinea-pig isolated colon.

The mechanical effects of 16-methyl analogues of PGE2, mainly 16,16-dimethyl PGE2, on circular and longitudinal muscles of the guinea-pig isolated proximal colon were investigated. In circular muscle strips, PGE2 100 nM produced an initial contraction followed by relaxation, while 16(R)-methyl PGE2 and 16,16-dimethyl PGE2 (1 nM - 1 microM) produced sustained contractions. In longitudinal muscle strips, PGE2 and 16-methyl analogues of PGE2 produced only contractions. The contractile responses of both muscle strips to 16,16-dimethyl PGE2 were not influenced by atropine or tetrodotoxin, indicating that these analogues act directly on the muscles, but were eliminated by the omission of extracellular Ca ions or in the presence of 1 mM lanthanum ions. However, verapamil, a Ca channel blocker, did not block the contractile response to the methyl analogues in circular muscle strips, although it completely inhibited the contractile response of longitudinal muscle strips. These results suggest that the contractile effect of 16-methyl analogues of PGE2 on the circular muscle may be due to an increased influx of Ca ions mainly via receptor-sensitive and partly voltage-sensitive Ca channels, while the contractile effect of the analogues on the longitudinal muscle may be due to an increase in influx of Ca ions via voltage-sensitive Ca channels.     

Effect of prostaglandin F2 alpha on propulsive activity of the isolated segmental colon of the guinea-pig.

The effects of prostaglandin F2alpha (PGF 2alpha) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated. Using experimental conditions under which spontaneous propulsive activity was negligible, PGF2alpha (5X10(-8)X1X10(-6)M), added to the bathing medium increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1X10(-7)g/ml). The contractions produced by PGF2alpha (5X10(-7) -1X10(-5)M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1X10(-7) g/ml). From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF2alpha may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Vasoactive intestinal polypeptide induces neurogenic contraction of guinea-pig ileum. Involvement of acetylcholine and substance P.

The effect and mode of action of vasoactive intestinal polypeptide (VIP), a peptidergic neuromodulator in the gastrointestinal nervous system, were investigated in isolated muscle strips of the guinea-pig ileum. VIP induced concentration-dependent (20 nM-1 microM) contractions of longitudinal ileal strips. TTX (1 microM), a mixture of atropine (3 microM) and spantide (30 microM), a mixture of atropine (3 microM) and omega-conotoxin GVIA (100 nM), somatostatin (60 nM) and dynorphin (100 nM) abolished the effect of VIP. In most cases a small relaxation became evident. Desensitization to substance P in the presence of atropine prevented VIP-induced contraction. A partial inhibition was observed in the presence of atropine (3 microM), spantide (30 microM), omega-conotoxin GVIA (100 nM), beta-endorphin (265 nM), met-enkephalin (1100 nM) and a mixture of spantide (30 microM) and omega-conotoxin GVIA (100 nM). The action of VIP was not significantly modified by guanethidine (3 microM) or hexamethonium (150 microM). In circular ileal strips VIP (10-300 nM) caused concentration-dependent relaxations through a direct myogenic effect. These results indicate that the VIP produced contractions of the guinea-pig ileum are exclusively neurally mediated and involve a cholinergic as well as a noncholinergic-nonadrenergic (NANC) pathway. It is concluded that besides acetylcholine (Ach) VIP releases the peptidergic transmitter substance P from postganglionic nerve fibers of myenteric plexus. Opioid peptides and somatostatin modulate the activity of cholinergic and peptidegic nerves in the guinea-pig ileum. The release of substance P appears to depend completely on N-type voltage sensitive calcium channels.     

Calcitonin gene-related peptide and capsaicin inhibit the circular muscle of the guinea-pig ileum

Calcitonin gene-related peptide (CGRP) is known to excite, through the release of acetylcholine, the circular muscle (CM) of the guinea-pig ileum in vitro. In the present experiments, the effect of rat CGRP was investigated on the CM of tetrodotoxin-treated, spontaneously active ileum preparations. CGRP (1-10 nM) caused concentration-dependent inhibition of both the amplitude and frequency of spontaneous CM contractions. Capsaicin (a sensory stimulant known to release CGRP from primary afferents) also inhibited CM activity. The effect of 1 microM capsaicin underwent rapid desensitization, indicating specific action on afferent structures, whereas a high concentration of the drug (33 microM) inhibited CM activity most probably on the smooth muscle itself.     

Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation.

Several nitric oxide (NO) effects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important effectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-d,l-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1-1000 microM) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 microM) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 microM) prevented NO effects, confirming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 microM), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 microM) prevented SNAP inhibitory effects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 microM) was ineffective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative effect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1-100 microM), inhibited cell proliferation. In addition, sub-effective concentrations of ODQ (100 nM) and TEA (10 microM) synergized in blocking SNAP antiproliferative effects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation.     

Nitric oxide induces muscular relaxation via cyclic GMP-dependent and -independent mechanisms in the longitudinal muscle of the mouse duodenum.

The aim of this study was to investigate, in mouse duodenum, the role of nitric oxide (NO) in the relaxation of longitudinal muscle evoked by nerve activation and the coupled action mechanism. Electrical field stimulation (EFS; 0.5 ms, 10-s train duration, supramaximal voltage, at various frequencies) under nonadrenergic noncholinergic conditions evoked muscular relaxation occasionally followed, at the higher stimulus frequencies, by rebound contractions. Inhibition of the synthesis of NO by N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM) virtually abolished the evoked relaxation. The relaxation was reduced also by apamin (0.1 microM) and by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a guanylyl cyclase inhibitor. The coadministration of apamin and ODQ produced additive effects on the responses to EFS. Sodium nitroprusside (0.1-100 microM) produced a concentration-dependent reduction of the phasic spontaneous activity and at the highest dose used suppressed phasic activity and induced muscular relaxation. These effects were tetrodotoxin and L-NAME resistant and were antagonized both by apamin and by ODQ. 8-Bromoguanosine 3',5'-cyclic monophosphate (0.1-100 microM) reduced in a concentration-dependent manner the spontaneous mechanical activity and at 100 microM suppressed the phasic activity and induced muscular relaxation, not antagonized by apamin. This study indicates that NO is the primary transmitter released by inhibitory nerves supplying the longitudinal muscle of mouse duodenum and that guanylate cyclase stimulation and opening of Ca(2+)-dependent K(+) channels are independent mechanisms working in parallel to mediate NO action.     

Different types of contractions in rat colon and their modulation by oxidative stress

The aim of this study was to investigate the modulation of in vitro rat colonic circular muscle contractions by dextran sodium sulfate (DSS)-induced inflammation and in spontaneous inflammation in HLA-B27 rats. We also examined the potential role of hydrogen peroxide (H(2)O(2)) in modulating excitation-contraction coupling. The muscle strips from the middle colon generated spontaneous phasic contractions and giant contractions (GCs), the proximal colon strips generated primarily phasic contractions, and the distal colon strips were mostly quiescent. The spontaneous phasic contractions and GCs were not affected by inflammation, but the response to ACh was suppressed in DSS-treated rats and in HLA-B27 rats. H(2)O(2) production was increased in the muscularis of the inflamed colon. Incubation of colonic muscle strips with H(2)O(2) suppressed the spontaneous phasic contractions and concentration and time dependently reduced the response to ACh; in the middle colon, it also increased the frequency of GCs. We conclude that H(2)O(2) mimics the suppression of the contractile response to ACh in inflammation. H(2)O(2) also selectively suppresses phasic contractions and increases the frequency of GCs, as found previously in inflamed dog and human colons.     

Functional coordination of motor activity in colonic smooth muscles in rat experimental model

Spontaneous and electrically-elicited motor activity was recorded by triple organ bath in rat segment-model preparation as display of excitation of local nerve networks and ascending or descending reflex pathways underlying contractile potency and functional coordination of colonic longitudinal and circular muscles. Spontaneous high-amplitude contractions, but not relaxations, appeared synchronously in both muscles. Electrical field stimulation applied to proximal or distal part of segments elicited both tetrodotoxin (0.1 microM)-sensitive local motor responses of the stimulated part and ascending or descending motor responses of the contralateral, nonstimulated part of the preparations. Contractions characterized the local response of longitudinal muscle. The circular muscle responded with relaxation followed by contraction. Synchronous ascending contractions and descending contraction of the longitudinal muscle and relaxation followed by contraction of the circular muscle were observed when the middle part of segments was stimulated, thus indicating that locally-induced nerve excitation propagated via intrinsic ascending or descending nerve pathways that could be synchronously coactivated by one and the same stimulus. The ascending motor responses were more pronounced and the motor responses of longitudinal muscle were expressed more than those of circular muscle suggesting an essential role of ascending reflex pathways and longitudinal muscle in the coordinated motor activity of colon.     

The vasoinhibitory activity of bovine chromogranin A fragment (vasostatin) and its independence of extracellular calcium in isolated segments of human blood vessels.

Endothelium-independent vasoconstrictor responses in isolated segments of human internal thoracic artery (ITA) and saphenous vein (SV) were used as a bioassay system for the vasoinhibitory activity of bovine chromogranin A (CGA). Preincubation with vasostatin (0.8 micrograms/ml), containing the N-terminal domain of CGA, (CGA1-76, CGA1-113 and CGA1-143ff), inhibited the contractile responses evoked by 80 mM K+, 2.6 microM noradrenaline (NA), or 65 nM endothelin-1 (ET-1) in Ca(2+)-free solution in SV but not in ITA. The results demonstrate a vasoinhibitory activity in vasostatin and show that there is a marked difference between the arterial and venous segments in the Ca2+ independent component of the inhibitory response. A vascular role for the N-terminal domain of CGA is indicated, presumably by inhibiting Ca2+ release from intracellular stores in the human vein but not the artery.     

Circumferential,not longitudinal,colonic stretch increases synaptic input to mouse prevertebral ganglion neurons

The relationship between longitudinal and circular muscle tension in the mouse colon and mechanosensory excitatory synaptic input to neurons in the superior mesenteric ganglion (SMG) was investigated in vitro. Electrical activity was recorded intracellularly from SMG neurons, and muscle tension was simultaneously monitored in the longitudinal, circumferential, or both axes. Colonic intraluminal pressure and volume changes were also monitored simultaneously with muscle tension changes. The results showed that the frequency of fast excitatory postsynaptic potentials (fEPSPs) in SMG neurons increased when colonic muscle tension decreased, when the colon relaxed and refilled with fluid after contraction, and during receptive relaxation preceding spontaneous colonic contractions. In contrast, fEPSP frequency decreased when colonic muscle tension increased during spontaneous colonic contraction and emptying. Manual stretch of the colon wall to 10-15% beyond resting length in the circumferential axis of flat sheet preparations increased fEPSP frequency in SMG neurons, but stretch in the longitudinal axis to 15% beyond resting length in the same preparations did not. There was no increase in synaptic input when tubular colon segments were stretched in their long axes up to 20% beyond their resting length. The circumferential stretch-sensitive increase in the frequency of synaptic input to SMG neurons persisted when the colonic muscles were relaxed pharmacologically by nifedipine (2 microM) or nicardipine (3 microM). These results suggest that colonic mechanosensory afferent nerves projecting to the SMG function as length or stretch detectors in parallel to the circular muscle layer.     

The effect of leukotriene D4 on the isolated stomach and colon of the rat

The effect of synthetic leukotriene D4 (LTD4) was evaluated on isolated gastric longitudinal or circular smooth muscle and distal colon of the rat. The concentrations of LTD4, 2.5 X 10(-10)M to 5 X 10(-7)M, evoked minimal to maximal contractile responses. In addition, selected prostaglandins were used to identify the mediator of LTD4-induced contraction of gastric smooth muscle. FPL 55712 inhibited LTD4-induced contractions of gastric longitudinal or circular muscle. Indomethacin inhibited only LTD4-induced contractions of the longitudinal muscle. A combination of FPL 55712 and indomethacin produced greater inhibition of LTD4-induced contractions of longitudinal muscle than either agent alone. However, the same combination of inhibitors showed no greater effect than FPL 55712 alone on LTD4-induced contractions of circular smooth muscle. Unlike PGI2, PGF2, PGA2, or PGD2, PGE2 evoked contraction of the longitudinal muscle and relaxation of the circular muscle of the stomach. The dissimilar effect of PGE2 in the two smooth muscle layers of the rat stomach may signify that PGE2 is the prostaglandin released by LTD4 from the longitudinal and circular gastric muscle. However, the opposing pharmacologic effects following LTD4-induced release of prostaglandins in the circular muscle of the stomach would preclude the appearance of an inhibitory effect of indomethacin in this tissue. In contrast, PGE2 and other prostaglandins contract gastric longitudinal muscle in response to LTD4. Thus, these studies clearly suggest that LTD4 has both a direct and indirect effect on gastric smooth muscle of the rat. Unlike the stomach, LTD4-induced contraction of the distal colon was not inhibited by indomethacin while FPL 55712 antagonized contractions. Thus, these findings indicate a differential mechanism of stimulation of rat gastrointestinal tissue by LTD4.     

Comparative effects of endothelin and phorbol 12-13 dibutyrate in rat aorta

The vasoconstrictive properties of endothelin (ET-1) and the protein kinase C activator, phorbol 12-13 dibutyrate (PDB) were comparatively investigated in isolated rat aorta. ET-1 (0.3-100 nM) and PDB (10 nM-3 microM) induced a slowly developing sustained contraction in endothelium denuded aorta. Maximal contractions induced by ET-1 and PDB were unaffected by diltiazem (10 microM). Substantial contraction to ET-1 (30 nM) and PDB (0.1 microM) remained in calcium-free medium. Contractions of ET-1 and PDB in calcium-free medium were unaffected by intracellular calcium depletion induced by phenylephrine. Following the response to ET-1 and PDB in a calcium-free medium, an additional sustained contraction was observed after calcium (2.5 mM) was added to the bath. The protein kinase C inhibitor, H7 (100 microM) was more potent in inhibiting contractions induced by phenylephrine and KCl than the ones elicited by ET-1 and PDB. The other protein kinase C inhibitors i.e. staurosporine (50 nM) and phloretin (100 microM) inhibited to a similar extent all the agonists tested. These results suggest that protein kinase C may play an important role in mediating the contraction to ET-1 in rat aorta.