Feline lower esophageal sphincter sling and circular muscles have different functional inhibitory neuronal responses

The lower esophageal sphincter (LES) has a circular muscle component exhibiting spontaneous tone that is relaxed by nitric oxide (NO) and a low-tone sling muscle that contracts vigorously to cholinergic stimulation but with little or no evidence of NO responsiveness. This study dissected the responses of the sling muscle to nitrergic innervation in relationship to its cholinergic innervation and circular muscle responses. Motor responses were induced by electrical field stimulation (EFS; 1-30 Hz) of muscle strips from sling and circular regions of the feline LES in the presence of cholinergic receptor inhibition (atropine) or NO synthase inhibition [NG-nitro-L-arginine (L-NNA)+/-atropine]. This study showed the following. First, sling muscle developed less intrinsic resting tone compared with circular muscle. Second, with EFS, sling muscle contracted (most at 50% by 5 Hz. Third, on neural blockade with atropine or L-NNA+/-atropine, 1) sling muscle, although predominantly influenced by excitatory cholinergic stimulation, had a small neural NO-mediated inhibition, with no significant non-NO-mediated inhibition and 2) circular muscle, although little affected by cholinergic influence, underwent relaxation predominantly by neural release of NO and some non-NO inhibitory influence (at higher EFS frequency). Fourth, the sling, precontracted with bethanecol, could relax with NO and some non-NO inhibition. Finally, the tension range of both muscles is similar. In conclusion, sling muscle has limited NO-mediated inhibition to potentially augment or replace sling relaxation effected by switching off its cholinergic excitation. Differences within the LES sling and circular muscles could provide new directions for therapy of LES disorders.     

Purinergic and nitrergic junction potential in the human colon

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 microM). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 microM), MRS 2179 (1 microM), and NF023 (10 microM). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).     

Different responsiveness of excitatory and inhibitory enteric motor neurons in the human esophagus to electrical field stimulation and to nicotine

To compare electrical field stimulation (EFS) with nicotine in the stimulation of excitatory and inhibitory enteric motoneurons (EMN) in the human esophagus, circular lower esophageal sphincter (LES), and circular and longitudinal esophageal body (EB) strips from 20 humans were studied in organ baths. Responses to EFS or nicotine (100 microM) were compared in basal conditions, after N(G)-nitro-l-arginine (l-NNA; 100 microM), and after l-NNA and apamin (1 microM). LES strips developed myogenic tone enhanced by TTX (5 microM) or l-NNA. EFS-LES relaxation was abolished by TTX, unaffected by hexamethonium (100 microM), and enhanced by atropine (3 microM). Nicotine-LES relaxation was higher than EFS relaxation, reduced by TTX or atropine, and blocked by hexamethonium. After l-NNA, EFS elicited a strong cholinergic contraction in circular LES and EB, and nicotine elicited a small relaxation in LES and no contractile effect in EB. After l-NNA and apamin, EFS elicited a strong cholinergic contraction in LES and EB, and nicotine elicited a weak contraction amounting to 6.64 +/- 3.19 and 9.20 +/- 5.51% of that induced by EFS. EFS elicited a contraction in longitudinal strips; after l-NNA and apamin, nicotine did not induce any response. Inhibitory EMN tonically inhibit myogenic LES tone and are efficiently stimulated both by EFS and nicotinic acetylcholine receptors (nAChRs) located in somatodendritic regions and nerve terminals, releasing nitric oxide and an apamin-sensitive neurotransmitter. In contrast, although esophageal excitatory EMN are efficiently stimulated by EFS, their stimulation through nAChRs is difficult and causes weak responses, suggesting the participation of nonnicotinic mechanisms in neurotransmission to excitatory EMN in human esophagus.     

Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.

Studies were performed in the opossum to define the role of the L-arginine-nitric oxide (NO) pathway in lower esophageal sphincter (LES) relaxation to swallowing and vagal stimulation in viv and intramural nerve stimulation in vitro. In vivo, L-NAME, a water soluble NO synthase (NOS) inhibitor, caused antagonism of LES relaxation due to reflex-induced swallowing. L-NAME (20 mg/kg i.v.) reduced the amplitude of swallow induced relaxation from 88% to 28%. LES relaxation due to electrical stimulation of peripheral end of decentralized vagus nerve was also antagonized. The effects of L-NAME were reversed by L-arginine, but not by D-arginine. L-NAME treatment did not antagonize LES relaxation to intravenous administration of isoproterenol. In vitro, NO and sodium nitroprusside (SNP) caused a decrease in the sphincter tone. The relaxing effect caused by NO and SNP was not antagonized by tetrodotoxin or omega-conotoxin. Inhibitors of NO synthase, L-NMMA and L-NNA, caused slight increase in the spontaneous resting LES tone and concentration-dependent antagonism of electrical field stimulation (EFS) induced LES relaxation. L-NNA (10(-4)M) abolished EFS induced LES relaxation at low frequencies (less than 5 Hz) and antagonized the relaxation to a value 20% of the control at 20 Hz. The antagonistic action of L-NMMA and L-NNA was unaffected by D-arginine but was reversed by L-arginine. The inhibitory effect of NO, SNP, or two other putative inhibitory neurotransmitters (VIP and CGRP) on the LES was not antagonized by L-NNA. These studies show that inhibitors of NO synthase selectively antagonize LES relaxation to all three modes of intramural inhibitory nerve stimulation including physiological swallowing. These studies suggest that the L-arginine-nitric oxide pathway is involved in physiological relaxation of the LES.     

P2Y1 receptors mediate inhibitory purinergic neuromuscular transmission in the human colon

Indirect evidence suggests that ATP is a neurotransmitter involved in inhibitory pathways in the neuromuscular junction in the gastrointestinal tract. The aim of this study was to characterize purinergic inhibitory neuromuscular transmission in the human colon. Tissue was obtained from colon resections for neoplasm. Muscle bath, microelectrode experiments, and immunohistochemical techniques were performed. 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) was used as a selective inhibitor of P2Y(1) receptors. We found that 1) ATP (1 mM) and adenosine 5'-beta-2-thiodiphosphate (ADPbetaS) (10 microM), a preferential P2Y agonist, inhibited spontaneous motility and caused smooth muscle hyperpolarization (about -12 mV); 2) MRS 2179 (10 microM) and apamin (1 microM) significantly reduced these effects; 3) both the fast component of the inhibitory junction potential (IJP) and the nonnitrergic relaxation induced by electrical field stimulation were dose dependently inhibited (IC(50) approximately 1 microM) by MRS 2179; 4) ADPbetaS reduced the IJP probably by a desensitization mechanism; 5) apamin (1 microM) reduced the fast component of the IJP (by 30-40%) and the inhibitory effect induced by electrical field stimulation; and 6) P2Y(1) receptors were localized in smooth muscle cells as well as in enteric neurons. These results show that ATP or a related purine is released by enteric inhibitory motoneurons, causing a fast hyperpolarization and smooth muscle relaxation. The high sensitivity of MRS 2179 has revealed, for the first time in the human gastrointestinal tract, that a P2Y(1) receptor present in smooth muscle probably mediates this mechanism through a pathway that partially involves apamin-sensitive calcium-activated potassium channels. P2Y(1) receptors can be an important pharmacological target to modulate smooth muscle excitability.     

Role of extrinsic innervation in modulating nitrergic transmission in the canine ileocolonic region

The human colon can dilate, often to life-threatening proportions. Our aim was to explore nitrergic mechanisms underlying colonic dilation in conscious dogs with enterically isolated ileocolonic loops either extrinsically innervated (n = 4) or extrinsically denervated (n = 4). We recorded phasic pressures in ileum and ileocolonic sphincter (ICS), colonic tone, compliance, and relaxation during ileal distension. By NADPH-diaphorase histochemistry, we assessed effects of extrinsic denervation and enteric isolation on nitrergic fibers. Extrinsic denervation increased phasic pressures in ileum, ICS, and colon and abolished ICS and colonic relaxation in response to ileal distension. The nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NNA) increased phasic pressures at all sites and ICS tone but did not abolish colonic relaxation during ileal distension in innervated loops. L-NNA reduced compliance and induced colonic high-amplitude propagated contractions in denervated loops. The NOS substrate donor L-arginine reversed effects of L-NNA. The number of NADPH-diaphorase fibers increased in both enterically isolated preparations. Nonnitrergic extrinsic nerve pathways mediate reflex colonic relaxation during ileal distension. Enteric isolation augments the number of NOS fibers, an effect not modified by extrinsic denervation.     

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

Background

Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.

Methods

Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.

Results

EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P < 0.01 all). In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz (P < 0.05 all), which was reversed by L-NNA to the level of control airways in the presence of L-NNA (P < 0.01 all). Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation (P < 0.05 all).

Conclusion

The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.     

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 μM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 μM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 μM), ODQ (10 μM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 μM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 μM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 μM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats

ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5'-nucleotidase inhibitor α,β-methylene adenosine 5'-diphosphate (AOPCP), 2) the A(2) receptor blocker ZM 241385, 3) the ADP P2Y(1) receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 μM, but not 100 μM, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 μM ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A(2) receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y(1) activation.     

Variability in the muscle composition of rat esophagus and neural pathway of lower esophageal sphincter relaxation

Several studies from our laboratory show that axial stretch of the lower esophageal sphincter (LES) in an oral direction causes neurally mediated LES relaxation. Under physiological conditions, axial stretch of the LES is caused by longitudinal muscle contraction (LMC) of the esophagus. Because longitudinal muscle is composed of skeletal muscle in mice, vagal-induced LMC and LES relaxation are both blocked by pancuronium. We conducted studies in rats (thought to have skeletal muscle esophagus) to determine if vagus nerve-mediated LES relaxation is also blocked by pancuronium. LMC-mediated axial stretch on the LES was monitored using piezoelectric crystals. LES and esophageal pressures were monitored with a 2.5-Fr solid-state pressure transducer catheter. Following bilateral cervical vagotomy, the vagus nerve was stimulated electrically. LES, along with the esophagus, was harvested after in vivo experiments and immunostained for smooth muscle (smooth muscle α-actin) and skeletal muscle (fast myosin heavy chain). Vagus nerve-stimulated LES relaxation and esophageal LMC were reduced in a dose-dependent fashion and completely abolished by pancuronium (96 μg/kg) in six rats (group 1). On the other hand, in seven rats, LES relaxation and LMC were only blocked completely by a combination of pancuronium (group 2) and hexamethonium. Immunostaining revealed that the longitudinal muscle layer was composed of predominantly skeletal muscle in the group 1 rats. On the other hand, the longitudinal muscle layer of group 2 rats contained a significant amount of smooth muscle (P < 0.05). Our study shows tight coupling between axial stretch on the LES and relaxation of the LES, which suggests a cause and effect relationship between the two. We propose that the vagus nerve fibers that cause LMC induce LES relaxation through the stretch-sensitive activation of inhibitory motor neurons.     

Mechanism of internal anal sphincter relaxation by CORM-1, authentic CO, and NANC nerve stimulation

The present studies compared the effects of CO-releasing molecule (CORM-1), authentic CO, and nonadrenergic noncholinergic (NANC) nerve stimulation in the internal anal sphincter (IAS). Functional in vitro experiments and Western blot studies were conducted in rat IAS smooth muscle. We examined the effects of CORM-1 (50-600 microM) and authentic CO (5-100 microM) and NANC nerve stimulation by electrical field stimulation (EFS; 0.5-20 Hz, 0.5-ms pulse, 12 V, 4-s train). The experiments were repeated after preincubation of the tissues with the neurotoxin TTX, the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), the selective heme oxygenase (HO) inhibitor tin protoporphyrin IX (SnPP-IX), the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), and SnPP-IX + L-NNA. We also investigated the effects of the HO substrate hematin (100 microM). CORM-1, as well as CO, produced concentration-dependent IAS relaxation, whereas hematin had no effect. TTX abolished and L-NNA significantly blocked IAS relaxation by EFS without any effect on CORM-1 and CO. ODQ blocked IAS relaxation by CORM-1, authentic CO, and EFS. SnPP-IX had no significant effect on IAS relaxation by CORM-1, CO, or EFS. The presence of neuronal nitric oxide synthase, HO-1, and HO-2 in IAS smooth muscle was confirmed by Western blot studies. CORM-1 and CO, as well as NANC nerve stimulation, produced IAS relaxation via guanylate cyclase/cGMP-dependent protein kinase activation. The advent of CORM-1 with potent effects in the IAS has significant implications in anorectal motility disorders with regard to pathophysiology and therapeutic potentials.     

Modular and functionally-different descending recto-anal motor pathways in a rat model

We evaluated the motor responses in recto-anal preparations obtained from rats, in terms of the excitation displayed by modules of nerve networks and descending distally directed pathways, when subjected to the mechanographic on-line technique, a partitioned organ bath, electrical stimulation (EFS, 0.8 ms, 5 Hz) and distension. EFS elicited modular contractions, which increased in amplitude distally, in circular muscle rings isolated from the proximal, middle or distal rectum. The modular responses of the internal anal sphincter or anal canal were relaxation or contraction, respectively. The application of EFS to the distal rectum induced a descending contractile response in the anal canal (5.24±0.34 mN), while distension by balloon evoked a descending response consisting of contraction (1.72±0.20 mN) followed by relaxation (3.42±0.24 mN). The responses were sensitive to tetrodotoxin. Atropine considerably depressed the contractions in all preparations. Whether or not atropine was present, L-NNA increased the excitatory responses, while L-arginine decreased the contractions and extended the relaxation of internal anal sphincter and anal canal. The results suggest that excitatory neurotransmission(s) expressed in the distal rectum dominate modular nerve networks. Functionally-different descending pathways are involved in the motor activity of the anal canal. Stimulatory cholinergic pathways are dependent on the electrically-induced excitation, and inhibitory nitrergic pathways are sensitive to distension of rectal wall.     

Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles

To determine whether densities ofcalmodulin (CaM) and CaM-binding proteins are related to phasic andtonic behavior of smooth muscles, we quantified these proteins in theopossum esophageal body (EB) and lower esophageal sphincter (LES),which represent phasic and tonic smooth muscles, respectively. Gelelectrophoresis, immunoprecipitation, Western blot, and hemagglutininepitope-tagged CaM (HA-CaM) overlay assay with quantitative scanningdensitometry and phosphorylation measurements were used. Total proteincontent in the two smooth muscles was similar (~30 mg protein/gfrozen tissue). Total tissue concentration of CaM was significantly(25%) higher in EB than in LES (P < 0.05).HA-CaM-binding proteins were qualitatively similar in LES and EBextracts. Myosin, myristoylated alanine-rich C kinase substrateprotein, Ca²⁺/CaM kinase II, and calponin contents werealso similar in the two muscles. However, content and total activity ofmyosin light chain kinase (MLCK) and content of caldesmon (CaD) werethree- to fourfold higher in EB than in LES. Increased CaM and MLCKcontent may allow for a wide range of contractile force varying fromcomplete relaxation in the basal state to a large-amplitude,high-velocity contraction in EB phasic muscle. Increased content ofCaD, which provides a braking mechanism on contraction, may furthercontribute to the phasic contractile behavior. In contrast, low CaM,MLCK, and CaD content may be responsible for a small range ofcontractile force seen in tonic muscle of LES.

     

Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus

The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.     

Coronary artery reperfusion: The ADP receptor P2Y<Subscript>1</Subscript> mediates early reactive hyperemia <Emphasis Type="BoldItalic">in vivo</Emphasis> in pigs

The physiological mechanisms that regulate reactive hyperemia are not fully understood. We postulated that the endothelial P2Y₁ receptor that release vasodilatory factors in response to ADP might play a vital role in the regulation of coronary flow. Intracoronary flow was measured with a Doppler flow-wire in a porcine model. 2-MeSADP (10^–5 M), ATP (10^–4 M) or UTP (10^–4 M) alone or as co-infusion with a selective P2Y₁ receptor blocker, MRS 2179 (10^–3 M) was locally delivered through the tip of a coronary angioplasty balloon. In separate pigs the coronary artery was occluded with the balloon for 10 min. During the first and tenth minutes of coronary ischemia, 2.5 ml of MRS 2179 (10^–3 M) was delivered distal to the occlusion in 8 pigs, 10 pigs were used as controls. MRS 2179 fully inhibited the 2-MeSADP-mediated coronary flow increase (P < 0.05) with no effect on UTP, indicating selective P2Y₁ inhibition. ATP-mediated flow increase was significantly inhibited by MRS 2179. During reactive hyperemia following coronary occlusion, flow increased by nearly sevenfold. MRS 2179, however, reduced the post-ischemic hyperemia by a mean of 46% during the period 1–2.5 min following balloon deflation (P < 0.05), which corresponds to peak velocity flow during reperfusion. In conclusion, MRS 2179, a selective P2Y₁ receptor blocker, significantly reduces the increased coronary flow caused both by 2-MeSADP and reactive hyperemia in coronary arteries. Thus, ADP acting on the endothelial P2Y₁ receptor may play a major role in coronary flow during post-ischemic hyperemia.     

Role of cGMP as a mediator of nerve-induced motor functions of the opossum esophagus

Stimulation of esophageal nerves produces biphasic relaxation of the lower esophageal sphincter (LES) and an off response of circular esophageal muscle. Previously, we proposed that cGMP mediates nerve-induced hyperpolarization of circular LES muscle but not LES relaxation. These experiments explore whether cGMP mediates LES relaxation or the off response. Strips of muscle from the opossum esophagus and LES were connected to force-displacement transducers, placed in tissue baths containing oxygenated Krebs solution at 37 degrees C, and stimulated by an electrical field. 1H-[1,2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of guanylyl cyclase, antagonized the off response, shortened its latency, and blocked the first phase of LES relaxation. ODQ also antagonized LES relaxation by exogenous nitric oxide (NO) but not relaxations by vasoactive intestinal polypeptide (VIP). Part of the nerve-induced LES relaxation and the off response appear to be mediated by the second messenger cGMP. These studies indicate that VIP-induced LES relaxation is not mediated by cGMP and therefore do not support the hypothesis that VIP produces LES relaxation by causing the generation of NO.     

Potassium channel diversity within the muscular components of the feline lower esophageal sphincter

We hypothesized that regional differences in electrophysiological properties exist within the musculature of the feline lower esophageal sphincter (LES) and that they may potentially contribute to functional asymmetry within the LES. Freshly isolated esophageal smooth muscle cells (SMCs) from the circular muscle and sling regions within the LES were studied under a patch clamp. The resting membrane potential (RMP) of the circular SMCs was significantly more depolarized than was the RMP of the sling SMCs, resulting from a higher Na+ and Cl- permeability in circular muscle than in sling muscle. Large conductance Ca2+-activated K+ (BKCa) set the RMP at both levels, since specific BKCa inhibitors caused depolarization; however, BKCa density was greatest in the circular region. A significant portion of the outward current was due to non-BKCa, especially in sling muscle, and likely delayed rectifier K+ channels (KDR). There was a large reduction in outward current with 4-aminopyridine (4-AP) in sling muscle, while BKCa blockers had a limited effect on the voltage-activated outward current in sling muscle. Differences in BKCa:KDR channel ratios were also manifest by a leftward shift in the voltage-dependent activation curve in circular cells compared to sling cells. The electrophysiological differences seen between the circular and sling muscles provide a basis for their different contributions to LES activities such as resting tone and neurotransmitter responsiveness, and in turn could impart asymmetric drug responses and provide specific therapeutic targets.     

Regional differences in L-type Ca2+ channel expression in feline lower esophageal sphincter

In humans and cats, muscle from the lower esophageal sphincter (LES) circular region exhibits greater spontaneous tone than LES sling muscle, whereas the sling muscle is much more responsive to cholinergic stimulation. Despite physiological and pharmacological evidence for the presence of L-type Ca2+ channel current (ICa,L) activity in LES circular muscle, the identity of this channel has not been demonstrated biochemically or electrophysiologically fingerprinted. Furthermore, there is no information on the channel's presence and role in the sling region of the LES. We hypothesized that regional differences in the expression of ICa,L between LES circular and sling muscles, if present, could contribute to the functional asymmetry observed within the LES. ICa,L expression was compared between circular and sling regions of the LES by Western blot analysis. The patch-clamp technique was used to study ICa,L. Muscle strip studies assessed ICa,L contribution to contractile activity. We found both protein expression of ICa,L and ICa,L density to be greater in LES circular muscle than sling muscle. ICa,L voltage- and time-dependent activation and inactivation curves were similar in cells from both regions. ICa,L blockade with nifedipine inhibited spontaneous tone and ACh-induced contractions only in circular muscle but was able to abolish depolarization (KCl)-induced contractions in both sling and circular muscles. In contrast, La3+ inhibited tone and ACh-induced contractions in muscles from both regions. Therefore, regional myogenic differences in ICa,L expression within the LES circular and sling muscle exist and provide one explanation for the differential contribution of sling and circular muscle to LES contractility.