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Biomedical subjects

K M Sanders

Publications and source records attributed to K M Sanders.

At least 19 recordsLinked to original sources
PubMed1992

Cromakalim and lemakalim activate Ca(2+)-dependent K+ channels in canine colon.

The effects of cromakalim (BRL 34915) and its (-) optical isomer, lemakalim (BRL 38227) on the activity of 265-pS Ca(2+)-activated K+ channels (BK channels) were examined in cell-attached and inside-out patches from canine colonic myocytes. In cell-attached patches lemakalim increased the open probability (Po) of BK channels. Mean NPo, where N is the number of channels per patch, at +50 mV increased from 0.08 to 0.26 (20 microM lemakalim). In inside-out patches, cromakalim and lemakalim increased channel NPo rapidly and reversibly. This increase in NPo was due to a shift in half-maximal activation. Glyburide (20 microM) prevented the increase in NPo caused by lemakalim in cell-attached patches and reversed the increase in NPo in inside-out patches. Under conditions where Ca(2+)-activated K+ channels were maximally activated, lemakalim failed to increase current or induce a second type of K+ channel activity. When tetraethylammonium (200 microM) was added to the pipette solution to block the BK channel half maximally, lemakalim also failed to induce a second type of channel. Adenosine triphosphate (1 or 2 mM) applied to the inner surface of inside-out patches had no effect on Po of BK channels. Finally, the effects of lemakalim on ensemble average currents, constructed from multiple openings of BK channels in cell-attached patches was found to successfully mimic the effects of the drug on whole-cell membrane currents. We conclude that cromakalim and lemakalim activate BK channels in canine colonic cells. Whether this action participates in the membrane hyperpolarization and the decrease in frequency and duration of slow waves produced by these compounds in intact colonic muscles remains to be investigated.

Adenosine Triphosphate↗
PubMed1992

Role of nitric oxide in non-adrenergic, non-cholinergic inhibitory junction potentials in canine ileocolonic sphincter.

1. Electrical field stimulation causes neurally-mediated relaxation of the ileocolonic sphincter that is due to activation of non-adrenergic and non-cholinergic (NANC) nerves. Recent studies have suggested that nitric oxide (NO) is the neurotransmitter that mediates relaxation. 2. Using intracellular recording techniques, we have tested whether NANC inhibitory junction potentials (i.j.ps) in the canine ileocolonic sphincter are also mediated by NO. 3. Electrical field stimulation elicited excitatory and inhibitory junction potentials: e.j.ps were blocked by atropine (10(-6) M) and tetrodotoxin (TTX; 10(-6) M); i.j.ps were also blocked by TTX and partially blocked by apamin (10(-6) M). I.j.ps were unaffected by atropine, phentolamine and propranolol (all at 10(-6) M). 4. The arginine analogues, L-NG-nitroarginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), decreased the amplitude of i.j.ps and L-arginine, but not D-arginine, partially restored the i.j.ps. 5. I.j.ps were also inhibited by oxyhaemoglobin (1%), but not by methaemoglobin. 6. Exogenous NO (10(-7) M to 3 x 10(-5) M) caused concentration-dependent hyperpolarizations that were similar in amplitude to the NANC nerve-evoked i.j.ps. Hyperpolarizations to NO were unaffected by L-NAME, but were blocked by oxyhaemoglobin. 7. Tetrodotoxin, L-NAME and oxyhaemoglobin all caused depolarization of resting membrane potential. 8. The specific guanosine 3':5'-cyclic monophosphate phosphodiesterase inhibitor, M&B 22948, caused hyperpolarization, increased the maximum level of hyperpolarization reached during i.j.ps, and increased the duration of i.j.ps. 9. These data further support the hypothesis that NANC neurotransmission in the ileocolonic sphincter is mediated by NO or an NO-releasing compound. The data also suggest that tonic release of NO, possibly from spontaneous firing of NANC nerves, may regulate resting membrane potential and tone in this sphincter.

Adrenergic Fibers↗
PubMed1992

Involvement of cyclic GMP in non-adrenergic, non-cholinergic inhibitory neurotransmission in dog proximal colon.

1. Nitric oxide (NO) may serve as a non-adrenergic, non-cholinergic (NANC) neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. We tested whether guanosine 3':5'-cyclic monophosphate (cyclic GMP) may serve as a second messenger in transducing the NO signal into inhibitory junction potentials (i.j.ps) and relaxation in the canine proximal colon. 2. The membrane permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8-Br-cyclic GMP) mimicked the effects of NO by hyperpolarizing cells near the myenteric border of the circular muscle layer and shortening slow waves in cells near the submucosal surface of the circular muscle layer. 8-Br-cGMP also inhibited spontaneous phasic contractions. 3. The specific cyclic GMP phosphodiesterase inhibitor, M&B 22948, hyperpolarized cells near the myenteric border and prolonged the duration of i.j.ps. M&B 22948 also inhibited phasic contractile activity. 4. Methylene blue failed to reduce significantly the amplitude and duration of i.j.ps and had variable effects on contractions. 5. Cyclic GMP levels were assayed in unstimulated muscles and in muscles exposed to exogenous NO and electrical field stimulation. Both stimuli hyperpolarized membrane potential, inhibited contractions, and elevated cyclic GMP levels. 6. Treatment of muscles with L-NG-nitroarginine methyl ester (L-NAME) increased spontaneous contractile activity and lowered cyclic GMP levels. The inhibitory effect of M&B 22948 on contractions was greatly reduced after muscles were treated with L-NAME. 7. These data support the concept that the effects of NANC nerve stimulation and NO (which may be one of the enteric inhibitory transmitters) may be mediated by cyclic GMP.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Spontaneous release of nitric oxide inhibits electrical, Ca2+ and mechanical transients in canine gastric smooth muscle.

1. In canine antrum, rhythmic electrical activity consists of a rapid upstroke phase followed by a plateau depolarization. In response to slow waves, cytosolic Ca2+ ([Ca2+]cyt) and tension increased. 2. Addition of sodium nitroprusside (SNP, 0.5 microM) decreased the amplitude of the plateau phase of slow waves without significant effects on the upstroke depolarization. SNP also inhibited changes in [Ca2+]cyt and tension associated with the plateau potential. SNP induced a negative chronotropic effect at concentrations above 0.1 microM. 3. Similar to the effects of SNP, illumination of muscles during slow waves with ultraviolet (UV) light caused premature repolarization. UV illumination is known to release NO in some tissues. 4. L-NG-monomethyl-arginine (L-NMMA, 300 microM), Methylene Blue (MB, 5 microM) and oxyhaemoglobin (oxy-Hb, 5 microM) increased the force of contractions. In contrast, L-arginine (L-Arg, 300 microM) decreased contractile force and antagonized the effects of L-NMMA. 5. During the upstroke phase, SNP caused a small reduction in [Ca2+]cyt and a large reduction in force, suggesting that SNP caused a decrease in Ca2+ sensitivity. 6. In muscles permeabilized by alpha-toxin, cyclic GMP (100 microM) and UV illumination inhibited Ca(2+)-induced contraction (at pCa 5.5). 7. These data suggest that NO or NO-related compounds are spontaneously released in gastric muscles. These agents have two effects on excitation-contraction coupling: (i) inhibition (directly and/or indirectly) of the voltage-dependent Ca2+ channels that participate in the plateau phase of slow waves, and (ii) reduction in the Ca2+ sensitivity of the contractile element.

Animals↗
PubMed1992

Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle.

1. Agonists known to increase cyclic AMP levels in gastrointestinal smooth muscles were studied in isolated circular muscles of the canine antrum to investigate the mechanisms of the inhibitory effects of these agents. 2. Muscles were electrically active, generating typical slow wave activity. Cytosolic Ca2+ ([Ca2+]cyt; measured by Indo-1 fluorescence) and tension increased in response to slow waves. 3. Stimulation by isoprenaline (via beta 2-receptors) or forskolin, in the presence or absence of acetylcholine, inhibited the plateau phase and reduced phasic [Ca2+]cyt and contractile responses. 4. Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), had similar effects to isoprenaline and forskolin. 5. Increases in the plateau phase of slow waves and the associated increases in [Ca2+]cyt and tension caused by direct activation of voltage-dependent Ca2+ channels by Bay K 8644 (0.1 microM) were also reduced by forskolin. 6. Isoprenaline and forskolin induced negative chronotropic effects, but VIP increased frequency. 7. At a given level of [Ca2+]cyt, contractions were greater under control conditions than in the presence of isoprenaline, VIP and CGRP, suggesting that part of the inhibition produced by these agents may be due to decreased Ca2+ sensitivity of the contractile apparatus. 8. Experiments performed on alpha-toxin-permeabilized muscles confirmed that cyclic AMP-dependent effects involve reduced Ca2+ sensitivity of the contractile apparatus. Addition of cyclic AMP (3-300 microM) caused a reduction in Ca(2+)-induced contraction at a constant level of Ca2+ (pCa 5.5). 9. These results suggest that increased cyclic AMP and probably subsequent activation of protein kinase A: (i) decrease [Ca2+]cyt and contraction by an inhibition of Ca2+ influx during slow waves, and (ii) decrease the sensitivity of the contractile apparatus to [Ca2+]cyt. The membrane effects might occur directly by inhibition of Ca2+ channels or indirectly by increasing the open probability of K+ channels which would tend to cause premature repolarization of slow waves.

Animals↗
PubMed1992

Dependence of electrical slow waves of canine colonic smooth muscle on calcium gradient.

1. The ionic dependence of the upstroke and plateau components of slow waves of canine colonic circular muscles was studied. 2. Reduced extracellular Ca2+ caused a decrease in the amplitude of the upstroke and plateau components, a decrease in the depolarization velocity, and a decrease in frequency. The reduction in the upstroke phase per 10-fold reduction in external Ca2+ was close to the value predicted by the Nernst relationship, suggesting that the membrane permeability to Ca2+ increases steeply during this phase. 3. Nifedipine (10(-9)-10(-6)) reduced the plateau component, but concentrations of 10(-6) M did not abolish the upstroke component. The data suggest that a nifedipine-resistant component of Ca2+ current may be involved in the upstroke. 4. Inorganic Ca2+ channel blockers (Mn2+ and Ni2+) blocked spontaneous slow waves at concentrations of 1.0 mM or less. 5. The upstroke component was more sensitive to Ni2+ than to Mn2+; a concentration of 0.040 mM-Ni2+ caused more than a 50% reduction in upstroke velocity. Ni2+ also reduced the plateau phase of slow waves. 6. The results suggest that the upstroke and plateau components of slow waves are dependent upon activation of voltage-dependent Ca2+ currents. The current responsible for the upstroke is partially resistant to dihydropyridines (at least at 10(-6) M). The current responsible for the plateau component is nifedipine-sensitive.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Upstroke component of electrical slow waves in canine colonic smooth muscle due to nifedipine-resistant calcium current.

1. Electrical slow waves of gastrointestinal smooth muscles are not abolished by organic Ca2+ channel blocking drugs, such as nifedipine or D600. These compounds reduce the amplitude and duration of the plateau phase, but the upstroke phase of slow waves persists. 2. Voltage clamp experiments were performed on isolated circular muscle cells from the canine proximal colon to characterize the dihydropyridine-resistant component of inward current. Inward currents were measured at 25 and 35 degrees C. The higher temperature increased the amplitudes of the transient and sustained phases of the inward current. The voltage dependence of activation and inactivation of the inward current was not significantly changed at 35 vs. 25 degrees C. 3. At 35 degrees C the transient phase of the inward current was reduced but not blocked by nifedipine (10(-6) M). The sustained phase was blocked by nifedipine. 4. The block by nifedipine was voltage dependent, increasing with depolarization. At voltages reached during the upstroke depolarization about 35% of the inward current persisted in the presence of nifedipine (10(-6) M). This may be sufficient inward current to sustain the upstroke depolarization in intact muscles. 5. Nifedipine caused a 20 mV negative shift in the voltage dependence of inactivation suggesting that dihydropyridines may preferentially bind to Ca2+ channels in an inactivated state. 6. Ni2+ (< 100 microM) significantly decreased the transient phase of inward current. A combination of Ni2+ (40 microM) and nifedipine (10(-6) M) blocked all of the inward current at 35 degrees C. Combination of nifedipine (10(-6) M) and Ni2+ (40 microM) blocked slow waves in intact muscles. 7. Bay K 8644 (10(-6) M) increased the amplitude of the transient and sustained components of inward current. On a percentage basis the increase in the sustained component was greater than the increase in the transient component with test potentials in the range of -50 to -20 mV. This may explain why Bay K 8644 preferentially increases the plateau component of slow waves vs. the upstroke component. 8. The findings of this study suggest that the nifedipine resistance of the upstroke depolarization could be due to the voltage dependence of the block of Ca2+ channels by dihydropyridines. Thus a single class of voltage-dependent Ca2+ channels could be responsible for the upstroke and plateau phases of slow waves.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Calcium oscillations in freshly dispersed and cultured interstitial cells from canine colon.

Spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were used to monitor rhythmicity in freshly dispersed and cultured interstitial cells (IC) from the canine colon. The frequency of oscillations and responses to a number of channel blockers, agonists, ionic substitutions, and temperature were similar in freshly dispersed and cultured cells. An increase in the amplitude of Ca2+ oscillations after 3-6 days in culture and an increase in the rate of decline of [Ca2+]i in cultured IC were two differences noted between freshly dispersed and cultured cells. The frequency and amplitude of oscillations were a function of extracellular Ca2+ concentrations, and oscillations were abolished when the transmembrane flux of Ca2+ was reduced by nicardipine, La3+, or removal of Ca2+ from the extracellular medium. Oscillations persisted in the presence of ryanodine and ouabain. Lowered temperatures or a reduction in the concentration of Ca2+ in the medium reduced the frequency of spontaneous oscillations. Carbachol and substance P caused a transient increase in [Ca2+]i. Substance P then abolished spontaneous events. ATP and calcitonin gene-related peptide increased the frequency of spontaneous activity. Vasoactive intestinal peptide caused a temporary delay in spontaneous oscillations when added to the medium. Results indicate that freshly dispersed and cultured IC may be useful in studies of the mechanisms of rhythmicity in the gastrointestinal system.

Adenosine Triphosphate↗
PubMed1992

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle.

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Animals↗
PubMed1992

Participation of fast-activating, voltage-dependent K currents in electrical slow waves of colonic circular muscle.

The plateau phase of electrical slow waves in phasic gastrointestinal muscles is critical for excitation-contraction coupling. The plateau appears to depend upon a balance between inward Ca2+ current and outward K+ currents that is sustained for several seconds. Voltage-dependent, non-Ca(2+)-dependent K currents were studied in canine colonic circular muscle cells using the whole cell patch-clamp technique. At room temperature, depolarization activated a slow outward current that showed little inactivation during 500 ms. Increasing the temperature to 37 degrees C significantly increased the rate of activation of voltage-dependent outward current. The onset of the outward current overlapped the transient inward Ca2+ current, suggesting that this K current may act as a brake on the upstroke depolarization of electrical slow waves in intact muscles. Voltage-dependent outward current was sustained for the duration of test pulses. This current balanced the sustained inward current that was also activated at physiological test potentials. The outward current evoked by test pulses positive to -20 mV inactivated by at least 50% within 500 ms. Half inactivation occurred at -36 mV. Voltage-dependent K current was reduced by 4-aminopyridine (4-AP; 1-5 mM), but difference currents obtained by subtracting currents elicited from holding potentials of -45 mV from currents obtained from holding potentials of -100 mV were not affected by 4-AP (1 mM). Studies were also performed on intact muscles to test the effects of 4-AP on electrical slow waves. 4-AP increased the amplitude and rate of rise of the upstroke potential and increased the amplitude and prolonged the plateau phase of slow waves. These data suggest that a rapidly activating, inactivating, voltage-dependent K current participates in electrical slow waves of colonic circular smooth muscles.

Animals↗
PubMed1992

Outward currents in longitudinal colonic muscle cells contribute to spiking electrical behavior.

Electrical events in longitudinal and circular muscles of the colon are different. Longitudinal muscles generate action potentials superimposed upon small depolarizations termed myenteric potential oscillations and circular muscles generate slow wave events that persist for several seconds. Differences between circular and longitudinal muscles may be related to the potassium channels these cells express. We have studied Ca(2+)-dependent and voltage-dependent K currents of isolated longitudinal cells with the whole cell patch-clamp technique. Test depolarizations positive to -40 mV yielded a transient inward current followed by a large sustained outward current. Blockade of the inward Ca2+ current reduced the amplitude of the outward current. Outward current was also reduced by tetraethylammonium (TEA; 1 mM), suggesting that a component of the outward current is Ca2+ dependent. After blockade of the Ca(2+)-dependent outward current, a voltage- and time-dependent component of outward current remained. The activation and inactivation properties and sensitivity to TEA and 4-aminopyridine (4-AP) were characterized. The voltage-dependent outward current in longitudinal cells had different properties than the voltage-dependent K currents in circular muscle cells (i.e., more negative inactivation, less sensitivity to 4-AP). TEA (1-5 mM) increased the amplitude and frequency of action potentials in intact longitudinal muscles; 4-AP (1 mM) had little effect on electrical activity of longitudinal muscles. The data suggest that differences in electrical behavior of the 2 muscle layers may be related to the expression of different species of K channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Negative-feedback regulation of excitation-contraction coupling in gastric smooth muscle.

The role of phosphatidylinositol (PI) turnover in excitation-contraction coupling was investigated in canine antral smooth muscle. Acetylcholine (ACh; 0.1-1 microM) transiently increased tissue levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and increased the amplitudes of the plateau phase of slow waves and associated Ca2+ transients and phasic contractions. ACh also increased basal concentrations of cytosolic Ca2+ ([Ca2+]c), but these changes were not associated with an increase in resting tension. ATP (0.3 mM) had similar effects on Ins(1,4,5)P3 levels, basal [Ca2+]c, and resting tension. However, in contrast to the effects of ACh, ATP transiently reduced the amplitude of the plateau phase of slow waves and reduced the amplitudes of associated Ca2+ transients and phasic contractions. We investigated the possibility that two products of PI turnover, diacylglycerol (DAG) and Ins(1,4,5)P3, might provide negative feedback to regulate Ca2+ entry during slow waves. 1) DAG is known to activate protein kinase C (PKC). Activation of PKC by phorbol 12,13-dibutyrate (PDBu, 0.5 microM) reduced the amplitude of the plateau phase of slow waves and corresponding Ca2+ transients and phasic contractions. Assay of PKC showed that ACh, ATP, and PDBu stimulated enzyme activity. 2) Ins(1,4,5)P3 is known to increase [Ca2+]c by release of Ca2+ from internal stores. Basal [Ca2+]c was also increased by elevated external K+, ionomycin, thapsigargin, or caffeine. Each of these compounds reduced the amplitude and duration of slow waves. Results suggest that products of PI turnover may provide negative-feedback control of Ca2+ influx during slow waves, tending to reduce the amplitude of phasic contractile activity in gastric muscles. Differences in responses to ACh and ATP can be explained by a G protein-dependent mechanism in which ACh suppresses the voltage dependence of Ca(2+)-activated K+ channels.

Acetylcholine↗
PubMed1992

Nonadrenergic, noncholinergic inhibition and rebound excitation in canine colon depend on nitric oxide.

Nonadrenergic, noncholinergic (NANC) nerves regulate slow waves along the submucosal border of the canine proximal colon. Experiments were performed to determine the role of nitric oxide (NO) in NANC responses. NANC responses are characterized by hyperpolarization and reduction in slow-wave amplitude and duration during the period of stimulation. This is followed by a "rebound" excitation (increase in amplitude and duration) of the slow wave immediately after the stimulus. These responses were blocked by L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase and by tetrodotoxin (TTX). Exogenous NO mimicked NANC responses, including the poststimulus rebound excitation. Responses to NO were unaffected by L-NAME or TTX. Responses to NANC nerve stimulation and NO were blocked by oxyhemoglobin but not by methemoglobin. Rebound excitation was reduced by pretreatment with indomethacin, suggesting that an eicosanoid may mediate this phase of NANC responses. Taken together, these data suggest that NO mediates NANC nerve responses in the proximal colon. NO appears to directly cause the inhibitory response, but the rebound response may depend on release of an eicosanoid.

Animals↗
PubMed1992

Electrical and mechanical effects of acetylcholine and substance P in subregions of canine colon.

Effects of acetylcholine (ACh) and substance P on the electrical and mechanical activities of the circular muscle layer of the canine proximal colon were studied. Because this muscle layer is bordered by two different pacemaker regions, responses from segments containing either a single pacemaker region or no pacemaker region were compared with responses of the complete muscle layer. Concentration-response relationships for ACh and substance P were similar between the various segments, suggesting that receptors for these agonists are expressed throughout the layer. The dominant contractile pattern induced by ACh and substance P in each segment was a 1- to 3-cycle/min rhythm. In a like manner, these agonists also elicited an electrical pattern in which a long-duration slow wave occurred one to three times per minute between short-duration slow waves. Low concentrations of nifedipine (0.01 microM) selectively antagonized the 1- to 3-cycle/min rhythm. In circular muscles with no pacemaker region, ACh (1 microM) caused depolarization, induced oscillations in membrane potential averaging 24 +/- 5 mV in amplitude and 2.9 +/- 0.9 cycles/min in frequency, and generated rhythmic contractions at the same frequency. This "interior" circular muscle was functionally innervated by cholinergic excitatory nerves. Exposure to ACh (1 microM) did not alter the conduction of slow waves through the thickness of the circular layer. In summary, the excitatory neurotransmitters, ACh and substance P, induce a dominant electrical and contractile rhythm throughout the circular muscle layer that is different from the spontaneous rhythms produced at either the myenteric or submucosal border.

Acetylcholine↗
PubMed1992

Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission.

Part of the regulation of gastrointestinal (GI) smooth muscles is provided by nonadrenergic noncholinergic (NANC) nerves. Stimulation of these nerves, either by field stimulation or via neural reflex pathways, elicits hyperpolarization of postjunctional smooth muscle membranes referred to as inhibitory junction potentials and relaxation. The transmitter(s) that mediate NANC inhibitory neural transmission have been a controversial topic for nearly 30 years. Recent evidence suggests that nitric oxide (NO) may serve as a NANC inhibitory transmitter in the GI tract. This hypothesis is supported by the following. 1) Immunohistochemical studies have shown that the enzyme necessary for NO synthesis is expressed in enteric neurons. In vitro studies of muscles from nearly all levels of GI tract have also shown that arginine analogues, which inhibit NO synthesis, reduce inhibitory effects of NANC neurotransmission. Effects of arginine analogues can be restored by addition of excess L-arginine, the substrate for NO synthesis. These data suggest that NO can be synthesized by enteric nerves. 2) Bioassays have demonstrated nerve-evoked release of a substance that has been identified as NO during NANC nerve stimulation. Oxyhemoglobin, known to bind to and sequester NO, also blocks NANC responses. These data suggest that NO is released into extracellular fluid during nerve stimulation. 3) Addition of NO causes rapid hyperpolarization of GI smooth muscle cells and relaxes muscles strips. These effects are similar to NANC nerve responses. NO and electrical field stimulation also increase tissue guanosine 3',5'-cyclic monophosphate, which may be the second messenger involved in NANC responses. 4) Removal of NO is easily accomplished by its rapid spontaneous breakdown in physiological solutions. 5) The pharmacology of NO and the NANC neurotransmitter in many preparations is similar, e.g., oxyhemoglobin blocks responses to NANC nerve stimulation and to exogenous NO. In summary, it would appear that many of the criteria necessary for NO to be considered a neurotransmitter have been satisfied.

Acetylcholine↗
PubMed1992

NADPH diaphorase and nitric oxide synthase colocalization in enteric neurons of canine proximal colon.

Considerable evidence has recently been presented that suggests that nitric oxide (NO) is a nonadrenergic noncholinergic (NANC) neurotransmitter in gastrointestinal tissues. One of the criteria that must be satisfied before this hypothesis can be accepted is that enteric neurons must be shown to contain the enzymatic apparatus necessary to synthesize NO. Specific antibodies have been developed for NO synthase (NOS) isolated from rat cerebellum, and studies have shown that NOS copurifies and colocalizes with NADPH diaphorase activity, a commonly used neural marker. We used antibodies raised against the cerebellar NOS to determine the distribution of NOS-like immunoreactivity (NOS-LI) in enteric neurons of the canine proximal colon. We also tested whether NADPH diaphorase staining would label the population of neurons containing NOS-LI in this species. A subpopulation of neurons in myenteric and submucosal ganglia displayed NOS-LI and were colabeled with NADPH diaphorase. Labeled neurons had morphological characteristics similar to the Dogiel type I morphology. Cryostat sections showed NOS-positive nerve trunks throughout the circular and longitudinal muscle layers, but a high density of NOS-LI was observed within the submucosal pacemaker region, as predicted from physiological studies. These studies provide the first morphological support for the hypothesis that NO serves as a NANC neurotransmitter in the canine colon. The study also shows that the NADPH diaphorase reaction provides a useful method to label cells with NOS-LI.

Amino Acid Oxidoreductases↗