Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission.

KEY POINTS: Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα+ cells that were closely associated with nNOS+ /VIP+ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were very similar. SK3 channel activation and inhibition caused faster/greater changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. ABSTRACT: Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with a high gene sequence identity to humans. Electrical field stimulation produced nitric oxide synthase (NOS)-dependent contractile inhibition in both muscles whereas P2Y1-dependent purinergic NMT was restricted to rectum. An additional NOS-independent, α-chymotrypsin-sensitive component was identified in the IAS consistent with vasoactive intestinal peptide-ergic (VIPergic) NMT. Microelectrode recordings revealed slow NOS-dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1-dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα+ /SK3+ cells were closely aligned with nNOS+ /VIP+ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2-14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was five times faster in rectum than in the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and the rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα+ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells.

ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter.

KEY POINTS: The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca2+ channels (CavL , encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. ABSTRACT: The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (encoded by Ano1) and voltage-dependent L-type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

Spatial organization and coordination of slow waves in the mouse anorectum.

The internal anal sphincter (IAS) develops tone and is important for maintaining a high anal pressure while tone in the rectum is less. The mechanisms responsible for tone generation in the IAS are still uncertain. The present study addressed this question by comparing the electrical properties and morphology of the mouse IAS and distal rectum. The amplitude of tone and the frequency of phasic contractions was greater in the IAS than in rectum while membrane potential (Em) was less negative in the IAS than in rectum. Slow waves (SWs) were of greatest amplitude and frequency at the distal end of the IAS, declining in the oral direction. Dual microelectrode recordings revealed that SWs were coordinated over a much greater distance in the circumferential direction than in the oral direction. The circular muscle layer of the IAS was divided into five to eight 'minibundles' separated by connective tissue septa whereas few septa were present in the rectum. The limited coordination of SWs in the oral direction suggests that the activity in adjacent minibundles is not coordinated. Intramuscular interstitial cells of Cajal and platelet-derived growth factor receptor alpha-positive cells were present in each minibundle suggesting a role for one or both of these cells in SW generation. In summary, three important properties distinguish the IAS from the distal rectum: (1) a more depolarized Em; (2) larger and higher frequency SWs; and (3) the multiunit configuration of the muscle. All of these characteristics may contribute to greater tone generation in the IAS than in the distal rectum.

Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells.

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCß) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+), and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCß were PDGFRα(+) cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα(+) cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI(-/-) mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. N(ω)-nitro-l-arginine (l-NNA) abolished responses during the first 20-30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.

Functional role of vasoactive intestinal polypeptide in inhibitory motor innervation in the mouse internal anal sphincter.

There is evidence that vasoactive intestinal polypeptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited, largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y1 receptor antagonist MRS2500 (1 µm) and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA; 100 µm) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (0.1-20 Hz, 4-60 s) and the associated changes in contractile and electrical activity measured in non-adrenergic, non-cholinergic conditions in the IAS of wild-type and VIP(-/-) mice. Electrical field stimulation gave rise to frequency-dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, an NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP6-28 (30 µm), absent in the VIP(-/-) mouse and mimicked by exogenous VIP (1-100 nm). Immunoreactivity for VIP was co-localized with neuronal nitric oxide synthase in varicose intramuscular fibres but was not detected in the VIP(-/-) mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in the IAS mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged period of anal relaxation.

Interstitial cells of Cajal in the cynomolgus monkey rectoanal region and their relationship to sympathetic and nitrergic nerves.

The morphology of interstitial cells of Cajal (ICC) in the circular muscle layer of the cynomolgus monkey internal anal sphincter (IAS) and rectum and their relationship to sympathetic and nitrergic nerves were compared by dual-labeling immunohistochemistry. Contractile studies confirmed that nitrergic nerves participate in neural inhibition in both regions whereas sympathetic nerves serve as excitatory motor nerves only in the IAS. Muscle bundles extended from myenteric to submucosal edge in rectum but in the IAS bundles were further divided into "minibundles" each surrounded by connective tissue. Dual labeling of KIT and smooth muscle myosin revealed KIT-positive stellate-shaped ICC (ICC-IAS) within each minibundle. In the rectum intramuscular ICC (ICC-IM) were spindle shaped whereas stellate-shaped ICC were located at the myenteric surface (ICC-MY). ICC were absent from both the myenteric and submucosal surfaces of the IAS. Nitrergic nerves (identified with anti-neuronal nitric oxide synthase antibodies or NADPH diaphorase activity) and sympathetic nerves (identified with anti-tyrosine hydroxylase antibody) each formed a plexus at the myenteric surface of the rectum but not the IAS. Intramuscular neuronal nitric oxide synthase- and tyrosine hydroxylase-positive fibers were present in both regions but were only closely associated with ICC-IM in rectum. Minimal association was also noted between ICC-IAS and cells expressing the nonspecific neuronal marker PGP9.5. In conclusion, the morphology of rectal ICC-IM and ICC-MY is similar to that described elsewhere in the gastrointestinal tract whereas ICC-IAS are unique. The distribution of stellate-shaped ICC-IAS throughout the musculature and their absence from both the myenteric and submucosal surfaces suggest that ICC-IAS may serve as pacemaker cells in this muscle whereas their limited relationship to nerves suggests that they are not involved in neuromuscular transmission. Additionally, the presence of numerous minibundles, each containing both ICC-IAS and nerves, suggests that this muscle functions as a multiunit type muscle.

Species dependent differences in the actions of sympathetic nerves and noradrenaline in the internal anal sphincter.

Excitatory motor innervation to the internal anal sphincter (IAS) of the monkey, the rabbit and mouse were compared. Contractile responses to electrical field stimulation of nerves (EFS, atropine 1 micromol L(-1) and N(omega)-nitro-L-arginine 100 micromol L(-1) present throughout) were examined in isolated strips of IAS. In the monkey IAS, EFS caused frequency dependent (1-30 Hz) contractions which were abolished by guanethidine (10 micromol L(-1)) or phentolamine (3 micromol L(-1)). The sympathetic neurotransmitter noradrenaline (NA) also caused concentration-dependent (10 nmol L(-1)-100 micromol L(-1)) contractions which were abolished by phentolamine revealing a small relaxation that was abolished by propranolol (3 micromol L(-1)). In contrast, EFS caused only relaxation of the mouse and rabbit IAS which was not affected by guanethidine. Furthermore, NA relaxed these muscles and relaxation was nearly abolished by combined addition of phentolamine and propranolol. In conclusion, the monkey IAS is functionally innervated by sympathetic nerves that contract the muscle via excitatory alpha-adrenergic receptors. In contrast, no significant motor function could be identified for sympathetic nerves in the rabbit or mouse IAS although adrenergic receptors linked to muscle inhibition are present. These data reveal species dependent differences in sympathetic motor innervation and suggest that some species are more appropriate than others as models for motor innervation to the human IAS.

Muscarinic M2 receptor stimulation of Cav1.2b requires phosphatidylinositol 3-kinase, protein kinase C, and c-Src.

This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 micromol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 micromol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 micromol/L), by the c-Src inhibitor PP2 (10 micromol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit Gbetagamma. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 micromol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.

Differential expression of P2X-purinoceptor subtypes in circular and longitudinal muscle of canine colon.

Adenosine triphosphate (ATP) mediates excitatory junction potentials through P2X receptors in many smooth muscles. However, relatively little is known about postjunctional intestinal P2X receptors. We examined the effect of exogenous ATP on circular and longitudinal myocytes of canine colon using the patch clamp technique at 32 degrees C. In both cell types, ATP induced inward currents (I(ATP)) at -70 mV in a concentration-dependent manner. The potency profile of ATP analogues in circular myocytes was: ATP approximately 2-methylthio-ATP > alpha,beta-methylene ATP, and that in longitudinal myocytes was: alpha,beta-methylene ATP approximately ATP > 2-methylthio-ATP. Pretreatment of circular myocytes with alpha,beta-methylene ATP inhibited the response to subsequent ATP, suggesting receptor desensitization. I-V relationships of I(ATP) were linear with inward rectification and E(rev) of -13 mV. I(ATP) at -70 mV was carried predominantly by Na+ as determined by shifts in E(rev) when extracellular Na+ was lowered. In RT-PCR, circular myocytes expressed mRNAs encoding P2X2, 3 and 4, while longitudinal myocytes expressed mRNAs for P2X3 and 5. P2X7 was absent in both cells. Fragments of each subtype were cloned and sequenced. We failed to clone P2X1 and P2X6 genes. Overall, different P2X receptor subtypes are expressed in circular and longitudinal canine colonic myocytes. Their activation produces non-selective cation currents that can depolarize and excite muscles of both layers.

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.

Endothelium-dependent relaxation and hyperpolarization in guinea-pig coronary artery: role of epoxyeicosatrienoic acid.

1. Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 microM N(omega)-nitro-L-arginine and 10 microM indomethacin. 2. ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H1-receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was significantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 microM). 3. The cytochrome P450 inhibitors proadifen (10 microM) and clotrimazole (10 microM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 microM) and 7-ethoxyresorufin (10 microM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 microM) and LEM (1 microM)-induced hyperpolarization. 4. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 microM), glibenclamide (10 microM) and BaCl2 (50 microM) had no significant effect on responses to ACh, AA and 11,12-EET. 5. IBTX (100 nM) significantly reduced both 11,12-EET (33 microM) and AA (30 microM) hyperpolarization without affecting the ACh (1 microM)-induced hyperpolarization. In contrast, 4-AP significantly reduced the ACh-induced hyperpolarization without affecting either AA or 11,12-EET-induced hyperpolarizations. 6. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO- and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

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.

Inhibition of nitric oxide synthesis reveals non-cholinergic excitatory neurotransmission in the canine proximal colon.

1. Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques. 2. Electrical field stimulation (EFS; 0.1-20 HZ) produced frequency-dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These responses were enhanced by NG-monomethyl-L-arginine (L-NMMA; 300 microM) and reduced by atropine (1 microM). The effects of L-NMMA were reversed by L-arginine (3 mM). All responses to EFS were abolished by tetrodotoxin (1 microM). 3. In the presence of atropine, phentolamine and propranolol (all at 1 microM; 'non-adrenergic, non-cholingergic (NANC) conditions'), EFS evoked frequency-dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post-stimulus excitation. 4. Under NANC conditions and in the presence of L-NG-nitroarginine methyl ester (L-NAME; 200 microM), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co-incubation with L-arginine (2 mM) and abolished by tetrodotoxin (1 microM). 5. In the presence of atropine (1 microM), EFS (5-20 Hz) caused frequency-dependent inhibition of electrical slow waves. In the presence of L-NAME (100 microM) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than 5 Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 microM). 6. Atropine-resistant contractions to EFS were enhanced by indomethacin (10 microM) and reduced or abolished by the non-selective NK1/NK2 tachykinin receptor antagonist D-Pro2, D-Trp7,9 SP, and by the selective NK2 receptor antagonist MEN 10,376 (10 microM).7. Exogenous tachykinins mimicked non-cholinergic excitatory electrical and mechanical responses. The rank order of potency for contraction was neurokinin A>neurokinin B>substance P, suggesting a predominance of the NK2 sub-type of tachykinin receptors on colonic smooth muscle cells. Low concentrations of neurokinin A also increased the amplitude and duration of electrical slow waves.8. These results suggest that: (i) in previous studies, non-cholinergic excitatory responses were masked by the simultaneous release of NO; (ii) non-cholinergic excitatory responses occur throughout the period of stimulation and are not manifest only as 'rebound' excitation; (iii) one or more tachykinins, possibly,acting via NK2 receptors, may mediate non-cholinergic excitatory responses.

Comparison of the actions of acetylcholine and BRL 38227 in the guinea-pig coronary artery.

1. The contractile and electrical responses to acetylcholine (ACh) in isolated segments of guinea-pig and rabbit coronary arteries were compared to those of the putative adenosine 5'-triphosphate (ATP)-dependent K+ channel opener, BRL 38227. 2. Both ACh and BRL 38227 produced concentration-dependent relaxation of vessel segments contracted with the H1-receptor agonist, 2-(2-aminoethyl)pyridine. 3. An IC90 of either vasodilator also produced 17-20 mV of hyperpolarization of the guinea-pig coronary artery. 4. Glibenclamide (1-35 microM) depolarized the guinea-pig coronary artery by 8-12 mV and antagonized BRL 38227- but not ACh-induced relaxation and hyperpolarization. 5. In the guinea-pig coronary artery, the K+ channel blockers phencyclidine (PCP, 100 microM), tetraethylammonium (TEA, 10 mM) and scorpion venom (8.7 micrograms ml-1) all significantly reduced ACh-induced relaxation and hyperpolarization whereas only PCP was an effective antagonist of both relaxation and hyperpolarization with BRL 38227. 6. Similar effects of glibenclamide and scorpion venom on ACh- and BRL 38227-induced relaxation were observed in the rabbit coronary artery. 7. Apamin (3.5 microM) was without effect on either the ACh- or BRL 38227-induced relaxation in the guinea-pig coronary artery. 8. In conclusion, the actions of BRL 38227 in coronary artery are compatible with its proposed effects on ATP-dependent K+ channels. In contrast, the results with ACh suggest that some step between the initial binding of ACh to endothelial muscarinic receptors and the final relaxation of the smooth muscle depends upon the opening of Ca(2+)-activated K+ channels.

Cyclic GMP-independent relaxation and hyperpolarization with acetylcholine in guinea-pig coronary artery.

1. The effects of acetylcholine (ACh) on membrane potential, relaxation and cyclic GMP levels were compared to the NO donor L-nitrosocysteine (Cys-NO) in segments of guinea-pig coronary artery. 2. ACh and Cys-NO produced concentration-dependent relaxations of muscles contracted with the H1 receptor agonist, 2-(2-aminoethyl)pyridine (AEP, 0.35 mM). The relaxation to ACh was unchanged in the presence of NG-monomethyl-L-arginine (L-NMMA; 350 microM) or indomethacin (3 microM). 3. Oxyhaemoglobin (HbO; 20 microM) alone or in combination with L-NMMA increased the EC50 for ACh-induced relaxation whereas relaxation with Cys-NO was almost completely abolished with HbO. 4. Scorpion venom (SV; 8.7 micrograms ml-1) increased the EC50 for relaxation with ACh but not Cys-NO. Combined L-NMMA, HbO and SV produced nearly complete abolition of ACh-induced relaxations. 5. Basal cyclic GMP levels (i.e., 20 pmol mg-1 protein) were significantly increased following addition of either ACh (190 pmol mg-1 protein) or Cys-NO (240 pmol mg-1 protein). L-NMMA significantly reduced the rise of cyclic GMP with ACh but not Cys-NO. In contrast, SV did not significantly reduce the rise in cyclic GMP produced with ACh. In the combined presence of L-NMMA and HbO neither ACh nor Cys-NO produced a significant increase in cyclic GMP levels. 6. ACh gave rise to significantly greater membrane hyperpolarization than Cys-NO both in the presence and absence of AEP. Combined L-NMMA and HbO did not reduce the amplitude of hyperpolarization with ACh. 7. These data indicate that some but not all of the actions of ACh in the coronary artery can be mimicked by the NO donor, Cys-NO, suggesting that ACh releases NO as well as a second hyperpolarizing factor (i.e., EDHF). Release of NO results in a large increase in tissue cyclic GMP levels and minimal change in membrane potential whereas release of EDHF results in a large membrane hyperpolarization which is independent of changes in tissue cyclic GMP levels. Both of these pathways appear to contribute to relaxation throughout the entire ACh concentration-relaxation relationship.

Frequency dependent alpha(2)-adrenoceptor mediated modulation of excitatory junction potentials in guinea-pig mesenteric artery.

Excitatory junctional potentials (EJPs) elicited with brief duration (10 s) electrical field stimulation of guinea-pig mesenteric arteries were nearly abolished at all frequencies by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 30 microM) but persisted following reserpinization. Suramin (100 microM) enhanced EJPs at 0.2-0.5 Hz responses and reduced them at 2-32 Hz. Phentolamine (1 microM) and yohimbine (0.1 microM) enhanced EJPs at 0.2-8 Hz but not at 16-32 Hz. Oxymetazoline (0.3 microM) reduced EJPs at 0.2-0.5 Hz but not at 1-32 Hz. Following reserpinization, EJPs were enhanced at 0.2-2 Hz but not at 4-32 Hz. Clonidine (0.1 microM) was without effect at all frequencies in control arteries but reduced EJPs at 0.2-2 Hz in reserpine-treated arteries. In conclusion, pre-junctional alpha(2)-adrenoceptors modulate ATP release during low frequency, brief duration sympathetic nerve stimulation.

Convergence of noncholinergic afferent neurons in the inferior mesenteric ganglion of the guinea pig.

The pattern of noncholinergic innervation of principal ganglionic neurons from the lumbar colonic nerve (LCN) and lumbar splanchnic nerve (LSN) in the inferior mesenteric ganglion (IMG) of the guinea pig was studied with intracellular techniques. Simultaneous stimulation of the LCN and LSN at maximum frequency (20 Hz) and submaximal stimulus voltages (2-4 V) led to summation of slow excitatory postsynaptic potential (EPSPs) indicating convergence of neural input. Summation was observed with submaximal (but not maximal) stimulation parameters in cells with either large or small amplitude maximum slow EPSPs suggesting that each neuron has an individual maximum capacity to depolarize in response to non-cholinergic transmitter substances. The study indicates that neurons originating at both peripheral and central sites converge onto principal ganglionic neurons, thus these ganglionic neurons must perform a significant integrative function in the IMG.

Cotransmission from sympathetic vasoconstrictor neurons: differences in guinea-pig mesenteric artery and vein.

Vasoconstrictor responses to electrical field stimulation (EFS, 0.2-32 Hz, 0.1 ms, 12 V, for 1 min) were measured in endothelium-denuded segments of guinea-pig mesenteric vein and compared to responses in mesenteric artery. The distribution of both tyrosine-hydroxylase-like immunoreactivity (TH-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) was also studied using anti-TH and anti-NPY antibodies. The effect of exogenous NPY (10 nM) on EFS (8 Hz, 0.3 ms, 12 V, for 1 min)-evoked overflow of noradrenaline (NA) was also studied using an HPLC technique with electrochemical detection. Veins responded with contractions at lower frequencies of stimulation than arteries. Prazosin (0.1 microM) abolished the EFS-evoked contractions in artery at 0.5-32 Hz and in vein at 0.2-1 Hz of stimulation. However, in vein, the contractile responses to EFS at 2-32 Hz of stimulation were only reduced by prazosin. Phentolamine (1 microM) abolished the responses to 0.5-4 Hz and reduced the responses to 8-32 Hz of EFS in artery. In vein, phentolamine (1 microM) abolished the responses to 0.2-1 Hz and facilitated the contractions elicited by 16-32 Hz. The NPY-receptor antagonist BIBP3226 (1 microM), in combination with phentolamine, abolished contractions in vein. Yohimbine (0.1 microM) abolished the responses to lower frequencies of stimulation in both artery (0.5-2 Hz) and vein (0.2-1 Hz). The responses to greater frequency stimulation were not affected by yohimbine in artery, and were facilitated in vein. Pre-treatment of animals for 24 h with reserpine abolished contractile responses to EFS in artery, whereas in vein, responses to 0.2-2 Hz were abolished while responses to 4-32 Hz were unchanged. Suramin (100 microM) or alpha,beta-methylene ATP (alpha,beta MeATP; 10-100 microM) treatment did not affect the contractile responses to EFS in either artery or vein. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 30 microM), even potentiated the responses to 2-16 Hz in vein. However, following resperine-treatment, both PPADS and suramin reduced the nerve-evoked contractions of vein. Either BIBP3226 (1 microM) alone or BIBP3226 in combination with PPADS or suramin abolished the contractile response to EFS in reserpine-treated veins. NPY (100 nM) produced significantly more contraction in vein than in artery (i.e., 93 +/- 2.5 versus 7 +/- 4% of the response to 70 mM KCl, respectively). NPY (10 nM) significantly reduced the NA overflow evoked by EFS at 8 Hz. Flat mount preparations and cryostat sections of both mesenteric artery and vein revealed that TH-LI and NPY-LI were co-localized in a dense network of fibers within the adventitial layer. In conclusion, NA exclusively mediates the contractile response to sympathetic nerve stimulation in guinea-pig mesenteric artery, whereas at least three neurotransmitters [i.e., NA, adenosine 5'-triphosphate (ATP) and NPY] are involved in the neural response of mesenteric vein.

Changes in neuromuscular transmission in the W/W(v) mouse internal anal sphincter.

BACKGROUND: Intramuscular interstitial cells of Cajal (ICC-IM) have been shown to participate in nitrergic neuromuscular transmission (NMT) in various regions of the gastrointestinal (GI) tract, but their role in the internal anal sphincter (IAS) is still uncertain. Contractile studies of the IAS in the W/W(v) mouse (a model in which ICC-IM numbers are markedly reduced) have reported that nitrergic NMT persists and that ICC-IM are not required. However, neither the changes in electrical events underlying NMT nor the contributions of other non-nitrergic neural pathways have been examined in this model. METHODS: The role of ICC-IM in NMT was examined by recording the contractile and electrical events associated with electrical field stimulation (EFS) of motor neurons in the IAS of wildtype and W/W(v) mice. Nitrergic, purinergic, and cholinergic components were identified using inhibitors of these pathways. KEY RESULTS: Under NANC conditions, purinergic and nitrergic pathways both contribute to EFS-induced inhibitory junction potentials (IJPs) and relaxation. Purinergic IJPs and relaxation were intact in the W/W(v) mouse IAS, whereas nitrergic IJPs were reduced by 50-60% while relaxation persisted. In the presence of L-NNA (NOS inhibitor) and MRS2500 (P2Y1 receptor antagonist), EFS gave rise to cholinergic depolarization and contractions that were abolished by atropine. Cholinergic depolarization was absent in the W/W(v) mouse IAS while contraction persisted. CONCLUSIONS & INFERENCES: ICC-IM significantly contributes to the electrical events underlying nitrergic and cholinergic NMT, whereas contractile events persist in the absence of ICC-IM. The purinergic inhibitory neural pathway appears to be independent of ICC-IM.