Movement based artifacts may contaminate extracellular electrical recordings from GI muscles.

BACKGROUND: Electrical slow waves drive peristaltic contractions in the stomach and facilitate gastric emptying. In gastroparesis and other disorders associated with altered gastric emptying, motility defects have been related to altered slow wave frequency and disordered propagation. Experimental and clinical measurements of slow waves are made with extracellular or abdominal surface recording. METHODS: We tested the consequences of muscle contractions and movement on biopotentials recorded from murine gastric muscles with array electrodes and pairs of silver electrodes. KEY RESULTS: Propagating biopotentials were readily recorded from gastric sheets composed of the entire murine stomach. The biopotentials were completely blocked by nifedipine (2 µmol L(-1) ) that blocked contractile movements and peristaltic contractions. Wortmannin, an inhibitor of myosin light chain kinase, also blocked contractions and biopotentials. Stimulation of muscles with carbachol increased the frequency of biopotentials in control conditions but failed to elicit biopotentials with nifedipine or wortmannin present. Intracellular recording with microelectrodes showed that authentic gastric slow waves occur at a faster frequency typically than biopotentials recorded with extracellular electrodes, and electrical slow waves recorded with intracellular electrodes were unaffected by suppression of movement. Electrical transients, equal in amplitude to biopotentials recorded with extracellular electrodes, were induced by movements produced by small transient stretches (<1 mm) of paralyzed or formalin fixed gastric sheets. CONCLUSIONS & INFERENCES: These data demonstrate significant movement artifacts in extracellular recordings of biopotentials from murine gastric muscles and suggest that movement suppression should be an obligatory control when monitoring electrical activity and characterizing propagation and coordination of electrical events with extracellular recording techniques.

Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle.

BACKGROUND AND PURPOSE: The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. EXPERIMENTAL APPROACH: We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. KEY RESULTS: The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents. CONCLUSIONS AND IMPLICATIONS: The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus.

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C.

Localized Ca(2+) transients resulting from inositol trisphosphate (IP(3))-dependent Ca(2+) release couple to spontaneous transient outward currents (STOCs) in murine colonic myocytes. Confocal microscopy and whole cell patch-clamp techniques were used to investigate coupling between localized Ca(2+) transients and STOCs. Colonic myocytes were loaded with fluo 3. Reduction in external Ca(2+) ([Ca(2+)](o)) reduced localized Ca(2+) transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca(2+) transients and STOCs showed increased coupling strength between Ca(2+) transients and STOCs when [Ca(2+)](o) was reduced. Gd(3+) (10 microM) did not affect Ca(2+) transients but increased STOC amplitude and frequency. Similarly, an inhibitor of Ca(2+) influx, 1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C (PKC) inhibitor, GF-109203X, also increased the amplitude and frequency of STOCs but had no effect on Ca(2+) transients. Phorbol 12-myristate 13-acetate (1 microM) reduced STOC amplitude and frequency but did not affect Ca(2+) transients. 4alpha-Phorbol (1 microM) had no effect on STOCs or Ca(2+) transients. Single channel studies indicated that large-conductance Ca(2+)-activated K(+) (BK) channels were inhibited by a Ca(2+)-dependent PKC. In summary 1) Ca(2+) release from IP(3) receptor-operated stores activates Ca(2+)-activated K(+) channels; 2) Ca(2+) influx through nonselective cation channels facilitates activation of PKC; and 3) PKC reduces the Ca(2+) sensitivity of BK channels, reducing the coupling strength between localized Ca(2+) transients and BK channels.

Muscarinic stimulation increases basal Ca(2+) and inhibits spontaneous Ca(2+) transients in murine colonic myocytes.

Localized Ca(2+) transients in isolated murine colonic myocytes depend on Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3)) receptors. Localized Ca(2+) transients couple to spontaneous transient outward currents (STOCs) and mediate hyperpolarization responses in these cells. We used confocal microscopy and whole cell patch-clamp recording to investigate how muscarinic stimulation, which causes formation of IP(3), can suppress Ca(2+) transients and STOCs that might override the excitatory nature of cholinergic responses. ACh (10 microM) reduced localized Ca(2+) transients and STOCs, and these effects were associated with a rise in basal cytosolic Ca(2+). These effects of ACh were mimicked by generalized rises in basal Ca(2+) caused by ionomycin (250-500 nM) or elevated external Ca(2+) (6 mM). Atropine (10 microM) abolished the effects of ACh. Pretreatment of cells with nicardipine (1 microM), or Cd(2+) (200 microM) had no effect on responses to ACh. An inhibitor of phospholipase C, U-73122, blocked Ca(2+) transients and STOCs but did not affect the increase in basal Ca(2+) after ACh stimulation. Xestospongin C (Xe-C; 5 microM), a membrane-permeable antagonist of IP(3) receptors, blocked spontaneous Ca(2+) transients but did not prevent the increase of basal Ca(2+) in response to ACh. Gd(3+) (10 microM), a nonselective cation channel inhibitor, prevented the increase in basal Ca(2+) after ACh and increased the frequency and amplitude of Ca(2+) transients and waves. Another inhibitor of receptor-mediated Ca(2+) influx channels, SKF-96365, also prevented the rise in basal Ca(2+) after ACh and increased Ca(2+) transients and development of Ca(2+) waves. FK-506, an inhibitor of FKBP12/IP(3) receptor interactions, had no effect on the rise in basal Ca(2+) but blocked the inhibitory effects of increased basal Ca(2+) and ACh on Ca(2+) transients. These results suggest that the rise in basal Ca(2+) that accompanies muscarinic stimulation of colonic muscles inhibits localized Ca(2+) transients that could couple to activation of Ca(2+)-activated K(+) channels and reduce the excitatory effects of ACh.

Intracellular calcium events activated by ATP in murine colonic myocytes.

ATP is a candidate enteric inhibitory neurotransmitter in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca(2+)-activated K(+) (SK) channels. Coupling between ATP stimulation and SK channels may be mediated by localized Ca(2+) release. Isolated myocytes of the murine colon produced spontaneous, localized Ca(2+) release events. These events corresponded to spontaneous transient outward currents (STOCs) consisting of charybdotoxin (ChTX)-sensitive and -insensitive events. ChTX-insensitive STOCs were inhibited by apamin. Localized Ca(2+) transients were not blocked by ryanodine, but these events were reduced in magnitude and frequency by xestospongin C (Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we have termed the localized Ca(2+) events in colonic myocytes "Ca(2+) puffs. " The P(2Y) receptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of Ca(2+) puffs. 2-MeS-ATP also increased STOCs in association with the increase in Ca(2+) puffs. Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, a P(2) receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca(2+) transients and the effects of 2-MeS-ATP on Ca(2+) puffs and STOCs were blocked by U-73122, an inhibitor of phospholipase C. Xe-C and ryanodine also blocked responses to 2-MeS-ATP, suggesting that, in addition to release from IP(3) receptor-operated stores, ryanodine receptors may be recruited during agonist stimulation to amplify release of Ca(2+). These data suggest that localized Ca(2+) release modulates Ca(2+)-dependent ionic conductances in the plasma membrane. Localized Ca(2+) release may contribute to the electrical responses resulting from purinergic stimulation.

Small conductance Ca2+-activated K+ channels are regulated by Ca2+-calmodulin-dependent protein kinase II in murine colonic myocytes.

1. Ca2+ regulates the activity of small conductance Ca2+-activated K+ (SK) channels via calmodulin-dependent binding. We investigated whether other forms of Ca2+-dependent regulation might control the open probability of SK channels. 2. Under whole-cell patch-clamp conditions, spontaneous openings of SK channels can be resolved as charybdotoxin-insensitive spontaneous transient outward currents (STOCs). The Ca2+-calmodulin-dependent (CaM) protein kinase II inhibitor KN-93 reduced the occurrence of charybdotoxin-insensitive STOCs. 3. The charybdotoxin-insensitive STOCs are related to spontaneous, local release of Ca2+. KN-93 did not affect spontaneous Ca2+-release events. 4. KN-93 and W-7, a calmodulin inhibitor, decreased the open probability of SK channels in on-cell patches but not in excised patches. 5. Application of autothiophosphorlated CaM kinase II to the cytoplasmic surface of excised patches increased the open probalibity of SK channels. Boiled CaM kinase II had no effect. 6. We conclude that CaM kinase II regulates SK channels in murine coloni myocytes. This mechanism provides a secondary means of regulation, increasing the impact of a given Ca2+ transient on SK channel open probability.

Review article: effects of the 5-HT3 receptor antagonist alosetron on neuromuscular transmission in canine and human intestinal muscle.

BACKGROUND: Currently, therapeutic treatments for irritable bowel syndrome fail to produce significant clinical results. We hypothesized that alosetron, a selective 5-HT3 antagonist, may provide symptomatic relief in irritable bowel syndrome patients through a decrease in the amplitude of gastrointestinal contractions. AIM: To determine the in vitro effect of alosetron on neuromuscular transmission in the canine and human jejunal and colonic muscularis externa. RESULTS: Alosetron diminished electrical field-stimulated (EFS) contractions recorded from muscles of the canine and human small and large intestines. Mechanistically, the diminished EFS response could be explained by the ability of alosetron to decrease the fractional release of 14C-choline radiolabelled acetylcholine evoked by EFS from human jejunal muscle. The inhibition of EFS contractions was not limited to atropine-sensitive events, as non-cholinergic excitatory EFS evoked contractions were also inhibited. Additionally, alosetron at high concentrations (> 30 microM) directly altered bethanechol stimulated contractions. CONCLUSION: Caution must be used in the interpretation of these data because significant alterations in EFS-induced contractions were only observed with large pharmacological concentrations of alosetron, and the response was not selective for cholinergically-mediated excitatory neuromuscular transmission.

Parallel pathways mediate inhibitory effects of vasoactive intestinal polypeptide and nitric oxide in canine fundus.

1. The gastric adaptation reflex is activated by the release of non-adrenergic, non-cholinergic (NANC) inhibitory transmitters, including nitric oxide (NO) and vasoactive intestinal polypeptide (VIP). The role of NO in this reflex is not disputed, but some investigators suggest that NO synthesis is stimulated by VIP in post-junctional cells or in nerve terminals. We investigated whether the effects of these transmitters are mediated by independent pathways in the canine gastric fundus. 2. VIP and NO produced concentration-dependent relaxation of the canine fundus. Nomega-nitro-L-arginine (L-NNA) reduced relaxation induced by electrical field stimulation (EFS; 0.5-8 Hz), but had no effect on responses to exogenous VIP and sodium nitroprusside (SNP, 10 microM). 3. Oxyhaemoglobin reduced relaxations produced by EFS and SNP. Oxyhaemoglobin also reduced relaxation responses to low concentrations of VIP (<10 nM), but these effects were non-specific and mimicked by methaemoglobin which had no effect on nitrergic responses. 4. A blocker of guanylyl cyclase, 1H-[1,2,4]oxidiazolo [4,3,-a]quinoxalin-1-one, (ODQ) inhibited responses to EFS, SNP and DETA/NONOate (an NO.donor), but had no effect on responses to VIP. cis-N-(2-phenylcyclopentil)-azacyclotridec-1en-2-amine monohydrochloride (MDL 12,330A), a blocker of adenylyl cyclase, reduced responses to EFS, VIP and forskolin, but did not affect responses to SNP. 5. Levels of cyclic GMP were enhanced by the NO donor S-nitroso-n-acetylpenicillamine (SNAP) but were unaffected by VIP (1 microM). The increase in cyclic GMP in response to SNAP was blocked by ODQ. 6. The results suggest that at least two transmitters, possibly NO and VIP, mediate relaxation responses in the canine fundus. NO and VIP mediate responses via cyclic GMP- and cyclic AMP-dependent mechanisms, respectively. No evidence was found for a serial cascade in which VIP is coupled to NO-dependent responses.

Dissociation between electrical and mechanical responses to nitrergic stimulation in the canine gastric fundus.

1. We examined the relationships between membrane potential, intracellular [Ca2+] ([Ca2+]i), and tension in muscles of the canine gastric fundus in response to nitrergic stimulation by NO donors and electrical field stimulation (EFS) of intrinsic enteric inhibitory neurons when adrenergic and cholinergic responses were blocked. 2. NO donors reduced [Ca2+]i and tension in a concentration-dependent manner. A close relationship was noted between these parameters. 3. In terms of the [Ca2+] vs. force relationship, relaxation responses to EFS differed from responses to NO donors. EFS resulted in smaller decreases in [Ca2+]i to produce a given relaxation compared with responses to NO donors. Thus, muscles stimulated with EFS were less sensitive to [Ca2+]i than muscles stimulated with exogenous NO. 4. When membrane potential, [Ca2+]i and tension were monitored simultaneously in the same muscles, a temporal dissociation was noted between the electrical responses and changes in [Ca2+]i and tension. Brief electrical responses were associated with more sustained changes in [Ca2+]i and tension. 5. Further dissociation between electrical and mechanical effects was noted. Changes in [Ca2+]i and tension caused by sodium nitroprusside and EFS were blocked by arginine analogues and by oxyhaemoglobin, but electrical responses were unaffected. 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, also blocked the effects of nitrergic stimulation on [Ca2+]i and tension, without affecting hyperpolarization. Thus, in the presence of continued hyperpolarization, the reductions in [Ca2+]i and tension caused by nitrergic stimulation were blocked. 6. Block of hyperpolarization in response to nitrergic stimulation with tetrapentylammonium chloride (TPEA) had relatively little effect on the [Ca2+]i and tension responses. Thus, hyperpolarization is not required for nitrergic effects on [Ca2+]i and tension. 7. In summary, reduction in [Ca2+]i and tension in response to nitrergic stimulation of the canine gastric fundus does not depend upon electrical hyperpolarization. Non-electrical mechanisms such as enhanced uptake of Ca2+ by the sarcoplasmic reticulum or reduction in the Ca2+ sensitivity of the contractile apparatus may be the primary mechanisms mediating nitrergic responses in these muscles.

Excitatory and inhibitory actions of vasopressin on colonic excitation-contraction coupling in dogs.

BACKGROUND & AIMS: Vasopressin, a circulating hormone and agent used to treat gastrointestinal bleeding, affects gastrointestinal motility. Both excitatory and inhibitory responses that may be caused by species differences or concentration-dependent effects of this hormone have been reported. This study examined the actions of vasopressin on the canine colon and studied the mechanism underlying the excitatory effects of vasopressin. METHODS: Intracellular microelectrodes and isometric force measurements were used to measure vasopressin responses in muscle strips. The patch-clamp technique was used to record arginine vasopressin activation of nonselective cation currents in isolated myocytes. RESULTS: Vasopressin (10(-12) to 10(-9) mol/L) increased electrical slow waves and enhanced phasic contractions. Atropine and tetrodotoxin did not alter the responses to vasopressin. In myocytes, vasopressin activated a nonselective cation conductance at concentrations that enhanced slow waves. Vasopressin (> 5 x 10(-8) mol/L) caused a tetrodotoxin- or NG-nitro-L-arginine methyl ester-insensitive inhibition of slow waves and contractile activity. A V1-receptor antagonist shifted the effects of vasopressin to higher concentrations. Immunohistochemistry showed expression of vasopressin-like immunoreactivity within the colonic wall, suggesting that local concentrations may exceed circulating levels. CONCLUSIONS: These data document the concentration dependence of the biphasic effects of vasopressin and provide a mechanism for the excitatory effects of vasopressin at physiological levels.

Role of Ca(2+)-activated K+ channels in electrical activity of longitudinal and circular muscle layers of canine colon.

The role of Ca(2+)-activated K+ channels (BK channels) in the canine colon was evaluated by testing the effects of charybdotoxin (ChTX) and tetraethylammonium on K+ currents of isolated myocytes and on electrical and mechanical activity of tissue strips. ChTX blocked Ca(2+)-activated outward current [IK(Ca)] in a dose- and voltage-dependent manner. No significant differences in IK(Ca) density, ChTX block, or Ca2+ sensitivity of BK channels were observed between circular and longitudinal myocytes. ChTX (100 nM) blocked 60% of current at +80 mV. Delayed rectifier current was not inhibited by 100 nM ChTX. In the absence of agonists, ChTX did not affect electrical or mechanical activity of circular muscle strips. In the presence of 10(-6) M BAY K 8644 or 10(-6) M acetylcholine, ChTX increased slow-wave duration and amplitude, induced membrane potential oscillations, and potentiated contraction. In unstimulated longitudinal muscle strips, ChTX depolarized the tissue, increased burst duration and spiking frequency, and resulted in an increase in contractions. These results indicate that BK channels are important regulators of colonic motility. In the longitudinal layer, BK channels are involved in setting membrane potential and determine excitability. In the circular layer, ChTX-sensitive channels do not participate in the in vitro basal electrical activity but limit the responses to excitatory agonists.

Relationship between nitric oxide and vasoactive intestinal polypeptide in enteric inhibitory neurotransmission.

Although considerable evidence suggests that NO serves as a neurotransmitter in gastrointestinal muscles, it is unlikely to be the only substance involved in enteric inhibitory neurotransmission. Vasoactive intestinal polypeptide (VIP) is known to be expressed by inhibitory motor neurons in the gut, and it appears to be co-localized with nitric oxide synthase (NOS) in a subpopulation of enteric neurons. These data suggest that NO and VIP may be parallel neurotransmitters. Others have suggested that VIP is the primary inhibitory transmitter, and it stimulates production of NO in smooth muscle cells. In this "serial cascade" model NO is a paracrine substance. We performed experiments on circular muscles and cells from the canine proximal colon to further test the idea that NO and VIP are parallel neurotransmitters and to determine the validity of the serial cascade model in these muscles. We found that NO-independent inhibitory effects were unmasked when excitatory and NO-dependent inhibitory responses were blocked. NO-independent inhibitory effects were reduced by alpha-chymotrypsin and blocked by tetrodotoxin. NOS- and VIP-like immunoreactivities were co-localized in enteric neurons and varicose fibers in the circular muscle layer. Similar to several other reports we found no evidence for a constitutive NOS in smooth muscle cells. Several aspects of the serial cascade model were not supported by our results: (i) the electrical and mechanical effects of VIP did not depend upon NO synthesis; (ii) VIP-induced changes in [Ca2+]i did not depend upon NO synthesis; and (iii) VIP did not cause the release of NO from canine colonic muscles. These results are consistent with the hypothesis that NO and VIP are co-transmitters, released in parallel from enteric inhibitory nerves.

Role of nonselective cation current in muscarinic responses of canine colonic muscle.

The mechanism of muscarinic excitation was studied in colonic muscle strips and isolated cells. In whole cell voltage-clamp studies performed at 33 degrees C utilizing the permeabilized patch technique, acetylcholine (ACh) reduced an L-type Ca2+ current. With K+ currents blocked, depolarization to positive potentials in the presence of ACh elicited outward current. Difference currents showed that ACh activated a voltage-dependent current that reversed at about -8 mV; this current (IACh) had properties similar to the nonselective cation conductance found in other smooth muscle cells. The reversal potential of IACh shifted toward negative potentials when external Na+ was reduced, and the inward current elicited at -70 mV decreased when external Na+ was reduced. IACh was facilitated by internal Ca2+. After the current was activated at a holding potential of -70 mV, depolarizations to -30 to 0 mV elicited influx of Ca2+ via voltage-dependent Ca2+ channels. After repolarization to the holding potential, a large inward tail current was observed. IACh was blocked by Ni2+ and Cd2+ at concentrations of 100 microM or less. Quinine (0.5 mM) also blocked IACh. With the use of the sensitivity of IACh to reduced external Na+ and divalent cations, the role of IACh in responses of intact muscles to ACh was examined. When external Na+ was reduced, ACh failed to increase slow-wave duration, and Ni2+ (50 microM) reversed the depolarization caused by ACh. These data suggest an important role for IACh in the electrical responses of colonic muscles. The contribution of IACh appears to prolong slow waves, which would allow greater entry of Ca2+ and increased force development.

Role of nitric oxide as an inhibitory neurotransmitter in the canine pyloric sphincter.

Experiments were performed to test the hypothesis that enteric inhibitory neurotransmission in pyloric muscles is mediated by NO. Junction potentials were recorded with intracellular microelectrodes from cells near the myenteric and submucosal surfaces of the circular muscle layer. Inhibitory junction potentials (IJPs) were apamin sensitive and were reduced by arginine analogues [NG-nitro-L-arginine-methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA)]. The effects of arginine analogues were reversed by L-arginine. Inhibition of IJPs unmasked excitatory JPs (EJPs) in the myenteric region and increased excitability of cells in the submucosal region. IJPs were also reduced by oxyhemoglobin. As with arginine analogues, reduction in IJPs increased EJP amplitude. Combination of L-NAME and oxyhemoglobin completely blocked IJPs, suggesting that NO, or an NO-containing compound, mediated the enteric inhibitory nerve responses. Exogenous NO hyperpolarized membrane potential, and these responses were also reduced by apamin. The magnitude of the responses to a given dose of NO was similar in cells of the myenteric and submucosal regions, suggesting that relatively smaller IJPs in submucosal cells may be due to a lower density of enteric inhibitory innervation in the submucosal region. The effects of NO were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and M & B 22948, a specific cGMP phosphodiesterase inhibitor, suggesting that the hyperpolarization response to NO may be mediated by enhanced production of cGMP. IJPs were also prolonged by M & B 22948. IJPs and NO disrupted normal electrical rhythmicity in cells in the myenteric region. This may provide a basis for inhibitory effects of enteric inhibitory nerve stimulation on sphincter pressure in pyloric canal in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of neural responses in the canine pyloric sphincter by opioids.

1. Regulation of excitatory and inhibitory junction potentials (e.j.ps and i.j.ps) by opioid peptides was studied in isolated muscle strips from the pyloric sphincter of the dog. 2. Methionine enkephalin (MetEnk; 10(-10) to 10(-6) M) and [D-Ala2, D-Leu5] enkephalin (DADLE; 10(-11) to 10(-7) M), a delta-specific opioid agonist, inhibited i.j.ps and e.j.ps recorded from cells in the myenteric and submucosal regions of the circular muscle layer. These compounds had no effect on resting potential or slow wave activity suggesting that the effects on junction potentials were not due to direct effects on smooth muscle cells. 3. MetEnk and DADLE caused similar effects on junction potentials in preparations in which the myenteric plexus was removed, suggesting that opioids inhibit pre-junctional effects on nerve fibres within the muscularis externa. 4. Inhibition of junction potentials by MetEnk and DADLE was blocked by approximately the same extent by naloxone (10(-6) M) and ICI 174,864 (10(-6) M), a delta-specific antagonist. 5. MetEnk and DADLE blocked a portion of the i.j.p. that was sensitive to arginine analogues; after treatment with N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), MetEnk and DADLE had no further effect on i.j.ps. These data suggest that opioids regulate nitric oxide-dependent neurotransmission. 6. Naloxone (10(-6) M) alone had no effect on i.j.ps elicited by short trains of electrical field stimuli. 7. I.j.p. amplitude was reduced after a period of conditioning stimulation (2 min, 30 Hz, 30 V). Naloxone blocked the post-stimulation inhibition. Repetitive stimulation at high frequencies (30 Hz) resulted in sustained hyperpolarization. Naloxone increased the amplitude of the hyperpolarization responses elicited by high frequency stimulation.8. These results show that e.j.ps and i.j.ps in the canine pylorus are inhibited by opioids. A portion of the inhibitory effects appears to be mediated via delta receptors.9. Although pyloric muscles are richly innervated by nerves containing opioid peptides, brief trains of stimuli do not appear to release concentrations of opioids that are effective in regulating junction potentials. Higher frequency stimulation (or longer durations of stimulation) appear to be necessary to release concentrations of opioids that are effective in modulating the amplitude of junction potentials.

Patterns of electrical activity and neural responses in canine proximal duodenum.

The patterns of electrical activity and neural inputs to the proximal duodenum between the pyloric sphincter and the sphincter of Oddi were studied in muscles of the dog. Smooth muscle cells in the most proximal region were electrically quiescent, but slow waves were recorded in all regions distal to the first few millimeters. Electrical activity was recorded from circular muscle cells near the myenteric and submucosal surfaces of the circular layer, and slow wave activity was similar in both regions. The nature of neural inputs was also characterized. With electrical field stimulation, responses in cells near the submucosal surface were predominantly excitatory junction potentials (EJPs); near the myenteric border responses were either inhibitory junction potentials (IJPs) or biphasic responses (i.e., small EJPs followed by IJPs). EJPs were blocked by atropine. IJPs were nonadrenergic and noncholinergic (NANC), and several experiments suggested that nitric oxide (NO), or a NO-releasing compound, serves as the inhibitory neurotransmitter in this region. Exogenous NO caused hyperpolarization of membrane potential. IJPs and the hyperpolarization response to NO were sensitive to apamin. These data describe the myogenic mechanisms and neurogenic apparatus that appear to regulate motility in the most proximal region of the duodenum.

Effect of cromakalim on the smooth muscle of the cat gastric antrum.

The effect of the K(+)-channel opener cromakalim (BRL 34915) on the electrical and contractile activity of the smooth muscle of the cat gastric antrum has been studied. Cromakalim induced a concentration-dependent inhibition of the contractions and shortening of the sustained partial repolarization phase of the plateau action potential. High concentrations of cromakalim produced hyperpolarization and shortening of the repolarization and depolarization phases of the plateau action potential. The K(+)-channel blockers 4-aminopyridine (10(-2) M) and tetraethylammonium (10(-2) M) decreased the effect of cromakalim on the phasic contractions, while glibenclamide (5 x 10(-5) M) completely abolished it. We suggested that the inhibitory effect of cromakalim on the electrical and contractile activity of the gastric antrum smooth muscle is due to the cromakalim-induced increase of the outward K(+)-current through glibenclamide-dependent K(+)-channels.

Character of the excitatory neurotransmission in dog terminal ileum.

In vitro experiments were performed on smooth muscle strips cut out longitudinally or circularly from the terminal ileum at about 5 cm from sphincter ileo-colocum of 3-4-month old and adult dogs. Studies were made on the postsynaptic responses of single smooth-muscle cells to transmural electrical stimulation of strips placed in sucrose gap, as well as on the contraction changes in response to field electrical stimulation of strips placed in organ bath. Field electrical stimulation elicited mainly excitatory responses of the muscle strips. Using cholino- and adrenoreceptor-antagonists and serotonergic receptor-blockers, we found that the majority of responses were cholinergic and a small part of them were alpha-adrenergic. Excitatory serotonergic responses were also obtained. The nerve nature of the responses was proved by tetrodotoxin. Single smooth-muscle cells gave predominantly excitatory postsynaptic responses to transmural stimulation of the smooth muscle strips. The pattern of both postsynaptic and contractile responses changed in the presence of the antagonists used. The present results suggest the predominance of cholinergic transmission in the contractile responses of the terminal ileum.

Spike activity of the muscle wall of the stomach remnant after gastric resection.

Chronic experiments have been carried out on dogs with implanted bipolar silver ball-shaped electrodes on the muscle wall of the stomach remnant after gastric resection. It has been found that in the first group of animals with subtotal gastrectomy (the frequency of the slow potentials during late periods after the resection increases and reaches the frequency of the corpus and antrum) the spike activity is represented by small groups of low-amplitude and low-frequency spike potentials. In the second group of animals for which the resection line passes at a distance of 1 to 1.5 cm distally from the boundary between the fundus and the corpus of the stomach (the frequency of the slow potentials increases to about 4 cpm even at the end of the first postoperational week), large groups of relatively high-amplitude and high-frequency spike potentials burst with the rhythmic slow potentials. The faster and better adaptation of the stomach remnant after sparse resection (2nd group of animals) is considered to be due to the existence of part of the pacemaker area in the stomach remnant. The spike activity characterizing the myoelectric complex is a prerequisite for the realization of the evacuation function by the stomach remnant.

Changes in the character of electrical and contractile activities of isolated fundus with functional loading (Billroth I).

A 2/3 resection of the stomach with restoration of the continuity by a Billroth I anastomosis was carried out on dogs and cats. At different times, following surgery, electrical activity of the fundus wall was recorded in vitro using pressure electrodes. Contractile activity of isolated fundus strips from these animals was also studied. Electrical activity of the isolated fundus of an intact (non resected) stomach was characterized by irregular low-amplitude slow waves of a sinusoidal type with a frequency of about 2 cpm, and in rare cases of about 4 cpm. Mechanical activity of the non-resected fundus was mainly tonic. In the isolated resected stomach (gastric remnant), a regular slow wave rhythm of about 4 cpm was established. Slow waves gradually increased in amplitude, achieving a plateau potential configuration. They were associated with low-amplitude contractions. The mechanical activity of the fundus after resection became mainly phasic. For the early establishment of this rhythm and configuration it was important to retain part of the pacemaker area of the stomach in the gastric remnant. In this case, electrical and contractile activities of the fundus became similar to those of the corpus and antrum, probably as a result of the functional loading of the fundus after resection.