Role of endopeptidase 3.4.24.16 in the catabolism of neurotensin, in vivo, in the vascularly perfused dog ileum.

1. The degradation of tritiated and unlabelled neurotensin (NT) following close intra-arterial infusion of the peptides in ileal segments of anaesthetized dogs was examined. 2. Intact NT and its catabolites recovered in the venous effluents were purified by chromatography on Sep-Pak columns followed by reverse-phase h.p.l.c. and identified by their retention times or by radioimmunoassay. 3. The half-life of neurotensin was estimated to be between 2 and 6 min. Four labelled catabolites, corresponding to free tyrosine, neurotensin (1-8), neurotensin (1-10) and neurotensin (1-11), were detected. 4. Neurotensin (1-11) was mainly generated by a phosphoramidon-sensitive cleavage, probably elicited by endopeptidase 24-11. 5. Two endopeptidase 3.4.24.16 inhibitors, phosphodiepryl 03 and the dipeptide Pro-Ile, dose-dependently potentiated the recovery of intact neurotensin. Furthermore, both agents inhibited the formation of neurotensin (1-10), the product that results from the hydrolysis of neurotensin by purified endopeptidase 3.4.24.16. In contrast, the endopeptidase 3.4.24.15 inhibitor Cpp-AAY-pAB neither protected neurotensin from degradation nor modified the production of neurotensin (1-10). 6. Our study is the first evidence to indicate that endopeptidase 3.4.24.16 contributes to the catabolism of neurotensin, in vivo, in the dog intestine.

Colocalization of inhibitory mediators, NO, VIP and galanin, in canine enteric nerves.

The colocalization of three putative inhibitory mediators of enteric nerves, vasoactive intestinal peptide (VIP), galanin (GAL) and nitric oxide synthase (nNOS), was examined in the myenteric plexus of canine antrum, intestine and colon. Many ileal and colonic neurons contained nNOS-immunoreactive (nNOS-IR) activity with some also containing VIP-IR; only a few neurons also contained GAL-IR. Ileal and colonic VIP-IR nerves often appeared to be interneurons innervating nNOS nerves. Many antral neurons contained VIP-IR with nearly all also containing GAL-IR. A few also contained nNOS-IR. The predominance of nNOS-IR neurons relative to VIP-IR and GAL-IR neurons in the ileal and colonic, but not the antral, myenteric plexus is consistent with NO being the primary inhibitory mediator in the intestine but not in the antrum.

Mechanism of noncholinergic excitation of canine ileal circular muscle by motilin.

In the isolated perfused canine ileal segment, exogenous motilin infused for 9 min, at concentrations from 10(-10) M and 10(-8) M, increased circular muscle motility concomitant with inhibiting tonic VIP release, maximum at 10(-8) M. Both effects increased with increasing motilin concentrations. Atropine 10(-7) M pretreatment did not alter these responses. Naloxone 10(-7) M pretreatment eliminated both the increase in motor activity and the inhibition of VIP levels. Thus the nonmuscarinic neural pathway responsible for motor activation by motilin probably involves the stimulation of release of opiates, which in turn inhibit the release of VIP. Reduction of tonic inhibition of the muscle by continuous VIP release may in part account for increases in motor activity induced by motilin.

Stimulation of circular muscle motility of the isolated perfused canine ileum: relationship to VIP output.

Perfusion of ileal segments with tetrodotoxin; opioids, Met-enkephalin and dynorphin; and alpha 2 adrenoceptor agonist, BHT920, increased motility concomitant with the decreased VIP output into the venous effluent reported previously. This suggested that increased motility resulted from release of the muscle from tonic inhibition when VIP output was reduced sufficiently. However, blockade of nicotinic receptors also reduced VIP output but did not induce motility. Thus release of myogenic activity from inhibition is not a sufficient explanation for increased motility and a further excitatory mediator is required. Field stimulation of nerves increased VIP output and delayed distal contractions, suggesting that VIP does participate in the canine ileal distal inhibition reflex.

Inhibitory and excitatory mechanisms of neurotensin action in canine intestinal circular muscle in vitro.

The effect of neurotensin on canine ileal circular muscle devoid of myenteric plexus was investigated using single and double sucrose gap techniques. Similar results were obtained with microelectrode techniques. Neurotensin caused a temperature-sensitive and dose-dependent biphasic response, an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase, usually consisting of spike discharge and tonic and phasic contractions, for which depolarization was not required. Neither response was affected by tetrodotoxin, phentolamine, propranolol, or atropine. The hyperpolarization was associated with decreased membrane resistance, blocked by 10(-7) M apamin, and converted to tonic depolarization by apamin (10(-6) M). Tachyphylaxis to neurotensin occurred when the stimulation interval was less than 20 min. After Ca2+ depletion, depolarization was observed instead of the hyperpolarization; this depolarization was not affected by nitrendipine and was gradually abolished with repetitive stimulation at 20-min intervals. When Ca2+ was present, nifedipine did not alter the hyperpolarizing phase of the response but inhibited spiking and blocked all contractions. The excitatory phase of the response was enhanced by Bay K-8644. Neuromedin N elicited a response identical with that of neurotensin. The responses of the two peptides were completely cross tachyphylactic. Inhibitory junction potentials were not affected by neurotensin tachyphylaxis. It is concluded that neurotensin and neuromedin N activate apamin-sensitive, calcium-dependent potassium channels in circular muscle, causing membrane hyperpolarization and inhibition of muscle contraction. Release of intracellular calcium is involved in the activation of these potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Sites of nitric oxide (NO) actions in control of circular muscle motility of the perfused isolated canine ileum.

In the isolated intra-arterially perfused canine ileum, N omega -nitro-L-arginine (L-NNA, 3 x 10(-4) M) increased tonic and phasic motor activity of the circular muscle. As has previously been shown, L-NNA enhanced contractions to electrical field stimulation at sites proximal to the serosal electrodes and converted initial relaxation when present at distal sites to contraction. L-NNA shifted the acetylcholine dose-response curve to the left and amplified the response to low-dose acetylcholine. Following L-NNA, addition of 10(-5) M sodium nitroprusside (NO donor) returned the tonic and phasic activity, the electrical field stimulation responses, and the acetylcholine dose-response curve to control values. Tetrodotoxin (TTX, 10(-6) M) increased tone (less than L-NNA) and abolished responses to both electrical field stimulation and motor activity induced by prior L-NNA. Subsequent L-NNA did not alter TTX-induced tonic motor responses. TTX also shifted the acetylcholine dose-response curve leftward and increased the responses to low-dose acetylcholine. After TTX, sodium nitroprusside returned the low-dose acetylcholine responses to control values and, after L-NNA, failed to restore them to control values. After L-NNA and TTX, sodium nitroprusside restored responses to low-dose acetylcholine to control values, Thus, removal of inhibition of the release of excitatory neurotransmitters, not removal of actions of NO on the muscle, accounted for the increases in tonic and phasic activity from L-NNA. Uninhibited release of excitatory transmitters augmented circular muscle responses to low-dose acetylcholine. TTX eliminated effects of excitatory transmitters, allowing exogenous NO to reduce low-dose acetylcholine contractions. No treatment affected the maximum responses to acetylcholine, produced by a contractile mechanism independent of muscle excitability and unaffected by exogenous NO or release of neurotransmitters.

Somatostatin excites canine ileum ex vivo: role for nitric oxide?

Isolated perfused segments of canine ileum have no spontaneous motor activity and release large quantities of vasoactive intestinal polypeptide (VIP) continuously. Somatostatin perfusion was shown to decrease VIP release, accompanied by increased contractions and amplification of responses to low-frequency electrical field stimulation. After perfusion of higher somatostatin concentrations, the VIP output did not recover but quiescence returned. The actions of somatostatin on motor activity were not modified by hexamethonium, slightly reduced by atropine, and markedly reduced by tetrodotoxin. Inhibition of VIP output was not the major determinant of motor activity in the ileum because 1) a second infusion of somatostatin had similar motor effects despite markedly reduced VIP output, 2) abolition of tonic VIP output did not prevent induction of motor activity by somatostatin, and 3) artificial restoration of VIP levels did not prevent or antagonize somatostatin-induced ileal contractions. In contrast, the increment in motor responses induced by somatostatin was not apparent after N omega-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase, but recovered after reversal by L-arginine. We conclude that the mode of somatostatin activation of intestinal motor activity involves reduced NO output, enhanced excitatory mediator action or release, a direct action on smooth muscle, and possibly inhibition of VIP output. Of these, reduced NO output plays the most important role.

Mechanisms of action of cholecystokinin in the canine gastrointestinal tract: role of vasoactive intestinal peptide and nitric oxide.

This study defined the cholecystokinin (CCK)/gastrin receptor subtypes at which CCK octapeptide (CCK8) and gastrin 17 (G17)act on motor functions of the intact canine gastrointestinal tract. In the antrum, studies of tachyphylaxis and effects of antagonists showed that i.a. G17 acted through CCKB receptors to activate contractions, whereas CCK8 acted through A and B receptor subtypes to produce contractions. In the duodenum, i.a. G17 caused dose-dependent inhibition of electrical field-stimulated contractions, apparently by release of nitric oxide [blocked by N-nitro-L-arginine (L-NNA) or NG-L-arginine methyl ester]. These inhibitory effects were abolished by YM022 (a CCKB antagonist) but not by L-364, 718 (a CCKA antagonist). However, i.a. CCK8 increased electrical field-stimulated contractions and L-364, 718 reversed this effect. In isolated perfused segments of distal intestine, CCK8 caused inhibition and excitation and released vasoactive intestinal peptide (VIP) into the venous effluent. CCK tetrapeptide and G17 had inconsistent effects. Excitation and VIP release were inhibited by L-364, 718. L-NNA potentiated excitatory responses and abolished inhibitory responses. Tetrodotoxin and atropine abolished and hexamethonium reduced excitatory responses to CCK8, but L-NNA restored contractions after atropine treatment. Hexamethonium or L-NNA (but not atropine) reduced VIP release; CCK8 still enhanced it. L-364, 718 abolished hexamethonium-resistant contractions and VIP release. Thus, CCK/gastrin peptides act on neural receptors in intact canine gastrointestinal tract. In antrum, activation of neural CCKA or CCKB receptors initiates contractions. In intestine, CCKA receptors at pre- and postjunctional sites in enteric nerves mediate acetylcholine and VIP release. CCKB receptors mediate release of an inhibitory mediator, apparently nitric oxide, from postjunctional sites.

Tachykinin-mediated increase in motility acts independently of vasoactive intestinal polypeptide release in the canine ileum.

Tachykinins induce motor activity in the canine ileum, and their mechanism of excitation may include inhibition of the release of a nonadrenergic, noncholinergic inhibitor, for which vasoactive intestinal polypeptide (VIP) is a candidate. Both substance P and neurokinin A produced a dose-dependent increase in ileal contractility with no significant change in VIP output. The highly selective NK1 agonist [Sar9, Met(O2)11]substance P and the highly selective NK2 agonist [Nle10]neurokinin A (4-10) also increased motor activity in the absence of any change in VIP released. These data suggest that the tachykinins produce motor activity in the canine ileum via a mechanism that does not involve changes in VIP output but may involve excitation through both NK1 and NK2 receptors.

Innervation of intestinal arteries by axons with immunoreactivity for the vesicular acetylcholine transporter (VAChT).

The presence of a cholinergic innervation of arterioles within the gut wall is suggested by pharmacological studies of nerve mediated vasodilatation, but attempts to identify nerve cells that give rise to cholinergic vasodilator fibres have yielded discrepant results. In the present work, antibodies to the vesicular acetylcholine transporter protein (VAChT) were used to investigate the relationships of immunoreactive nerve fibres to submucosal arterioles. Comparison was made with cerebral arteries, which are known to be cholinergically innervated. Double labelling immunohistochemical techniques revealed separate VAChT and tyrosine hydroxylase (TH) immunoreactive (IR) fibres innervating all sizes of arteries of the submucosa of the stomach, ileum, proximal colon, distal colon and rectum as well as the cerebral arteries. Arterioles of all digestive tract regions had greater densities of TH-IR innervation than VAChT-IR innervation. In the ileum, double labelling for VAChT-IR and VIP-IR or calretinin-IR showed more VAChT-IR than either VIP-IR or calretinin-IR fibres. Calretinin-IR and VAChT-IR were colocalised in a majority of calretinin-IR axons, but VIP-IR and VAChT-IR were not colocalised. All calretinin-IR nerve cells in submucous ganglia were immunoreactive for choline acetyltransferase, but only 1-2% of VIP-IR nerve cells were immunoreactive. Extrinsic denervation of the ileum did not alter the distribution of VAChT-IR fibres, but it eliminated TH-IR fibres. Removal of myenteric ganglia (myectomy) did not alter the distribution of fibres with VAChT or TH-IR. This work thus provides evidence for cholinergic innervation of intrinsic arterioles throughout the digestive tract and indicates that the fibres in the small intestine originate from submucosal nerve cells.

Local, exendin-(9-39)-insensitive, site of action of GLP-1 in canine ileum.

Glucagon-like peptide-1 (GLP-1) modulates glucose levels following a meal, including by inhibition of gastric emptying and intestinal transport. Intra-arterial injection of GLP-1 into the gastric corpus, antrum, or pylorus of anesthetized dogs had no effect on the contractile activity of the resting or neurally activated stomach. GLP-1 injected intra-arterially inhibited intestinal segments when activated by enteric nerve stimulation but not by acetylcholine. Isolated ileum segments were perfused intra-arterially, instrumented with strain gauges to record circular muscle activity and with subserosal electrodes to stimulate enteric nerves. GLP-1 caused concentration-dependent inhibition of nerve-stimulated phasic but not tonic activity. This was absent during TTX-induced activity and partly prevented by N(G)-nitro-L-arginine. Exendin-(9-39), the GLP-1 antagonist, had no intrinsic activity and did not affect the actions of GLP-1. Capsaicin mimicked the effects of GLP-1 and may have reduced the effect of subsequent GLP-1. GLP-1 may mediate paracrine action on afferent nerves in the canine ileal mucosa using an unusual receptor.

The tachykinin receptors inducing contractile responses of canine ileum circular muscle.

This study sought to determine which tachykinin receptors were involved in contractile responses of circular muscle to tachykinins infused into isolated segments of canine ileum. Selective agonists for neurokinin receptors NK1 and NK2 as well as for substance P (SP) and neurokinin A (NKA) were infused, and selective antagonists against NK1, NK2, and NK3 receptors were tested. The responses to a submaximal concentration of NKA were reduced by a selective NK2 antagonist, SR-48968, and abolished by a combination of this antagonist with an NK1 antagonist, either CP-96,345 or RP-67580. The selective NK2 agonist, [Nle10]NKA-(4-10), had low potency. We concluded that NKA acted on typical NK2 receptors and that is action was potentiated by its additional action on NK1 receptors. Neither the contractile responses to SP nor those to [Sar9,Met(O2)11]SP given in submaximal concentrations were inhibited by CP-96,345 or RP-67580, either alone or together with SR-48968. Indeed, the two NK1-selective antagonists potentiated responses to the selective NK1 agonist, [Sar9,Met(O2)11]SP, an effect attributed to previously demonstrated prejunctional inhibitory action of the agonist. The selective NK3 agonist, succinyl-[Asp6,N-Me-Phe8]SP-(6-11), was not effective as a contractile agent, even after block of nitric oxide synthase with N omega-nitro-L-arginine. The selective NK3 antagonist, R-487, was also ineffective in blocking responses to SP. Studies with an antagonist to H1 histamine receptors suggested that contractile actions of SP did not involve histamine release from mast cells. We concluded that, in addition to typical NK1 and NK2 receptors activated by NKA and a prejunctional inhibitory receptor activated by SP and [Sar9,Met(O2)11]SP, another tachykinin receptor existed on canine ileum to initiate contractions. It is not a typical NK1, NK2, or NK3 receptor.

Role of nitric oxide-related inhibition in intestinal function: relation to vasoactive intestinal polypeptide.

This study examined the role of nitric oxide (NO) in tonic inhibition of motor activity in isolated, perfused canine ileal segments. Brief addition of N omega-nitro-L-arginine methyl ester (L-NAME) to the perfusate caused, after a delay, a concentration-dependent persistent increase in tonic and phasic activity of circular muscle. This increased motor activity was prevented or reversed by addition of L- but not D-arginine to the perfusate. Removal of Ca2+ or addition of 10(-7) M omega-conotoxin (GVIA) to the perfusate markedly reduced this response. The motor activity induced by L-NAME was accompanied by loss of distal inhibition and enhanced excitation to low-frequency field stimulation. L-NAME infusion significantly reduced tonic vasoactive intestinal polypeptide (VIP) output, sodium nitroprusside increased VIP output, but L-arginine infusion did not restore VIP output. Atropine (10(-7) M) and/or hexamethonium (10(-4) M) reduced the motor response to L-NAME by 75%. Atropine reduced and hexamethonium nearly abolished VIP output. We conclude that there is tonic Ca(2+)-dependent NO output from perfused intestinal segments dependent on nerves with N-Ca channels, that NO acts to inhibit muscle directly and by inhibiting release of excitatory mediators, and that this output is the primary inhibitory determinant of contractile activity.

Identification of mechanisms and sites of actions of mu and delta opioid receptor activation in the canine intestine.

Perfusion with ([N-Me-Phe3,D-Pro4]morphiceptin (PL017)), [D-Pen2,5]enkephalin (DPDPE) and MEt5 and Leu5 enkephalin induced circular muscle contractions and decreased immunoreactive vasoactive intestinal polypeptide (VIP) venous output in canine ileal segments. Motility and VIP responses to PL017 were abolished by the mu antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) and unchanged by the delta antagonist ICI 174,864 ([N,N-dially-Tyr1,Aib2,3]Leu-enkephalin) which abolished DPDPE motility and VIP responses. The VIP response to DPDPE was unchanged by CTAP, which reduced motility responses, suggesting a DPDPE interaction with endogenous mu opioids, at a mu/delta(complexed) receptor. ICI 174,864 abolished Met5 and Leu5 enkephalin motility responses and Leu5 enkephalin VIP responses while CTAP was ineffective on Leu5 enkephalin motility responses or on both enkephalin VIP responses. CTAP increased Met5 enkephalin motility responses suggesting mu actions to inhibit excitatory nerves. ICI 174,864 reduced Met5 enkephalin VIP output decrements requiring CTAP addition for abolition, suggesting actions at mu/delta(complexed) receptors. Inhibition of nitric oxide synthase with N-omega-L-arginine methyl ester (L-NAME) abolished delta opioid and reduced by 30% mu opioid motility responses, leaving the VIP response intact. Hexamethonium and atropine abolished tonic VIP output, leaving intact motility responses to PL017 and DPDPE. Subsequently L-NAME eliminated delta opioid and reduced by 1/3 mu opioid motility responses. All opioids reduced the NO-mediated IJPs in myenteric plexus-free ileal circular muscle. Thus mu or delta opioids inhibit both NO and VIP release but removal of NO, not VIP, disinhibits circular muscle motility.

Pharmacological role and degradation processes of neuromedin N in the gastrointestinal tract: an in vitro and in vivo study.

Neuromedin N (NN) induced a concentration-dependent contraction (ED50 = 2.3 +/- 0.2 microM) of the isolated longitudinal smooth muscle from guinea pig ileum. This effect was drastically enhanced (ED50 = 0.06 microM) by the aminopeptidases M and B inhibitor bestatin (10 microM), which elicited a 40-fold increase in NN potency. HPLC analysis indicated that the main NN catabolite generated by membranes from guinea pig longitudinal smooth muscle homogenate corresponded to des-Lys1-NN, which results from removal of the N-terminal lysyl residue of NN. The fact that the formation of des-Lys1-NN was fully prevented by bestatin (10 microM) further supports the involvement of aminopeptidases in NN degradation. We examined the catabolic fate of NN in vivo in the vascularly perfused dog ileum. Bolus administration or continuous infusion of the peptide led to rapid disappearance of NN. This was prevented by prior treatment of ileal segments with bestatin (10 microM) but not with arphamenine B (0.5 microM), which indicated that aminopeptidase M but not aminopeptidase B participated in NN proteolysis in vivo. We showed that 1 and 10 nmol NN trigger the release of 28 +/- 5 and 59 +/- 1 pmol, respectively, of endogenous vasoactive intestinal polypeptide-like immunoreactivity after infusion in the vascularly perfused dog ileum. This release was virtually doubled by prior treatment with 10 microM bestatin but not with 0.5 microM arphamenine B. Altogether, our data indicate that aminopeptidase M is largely responsible for NN degradation in vitro and in vivo in the gastrointestinal tract and could be considered the physiological inactivator of NN in the gut.

Locations and molecular forms of PACAP and sites and characteristics of PACAP receptors in canine ileum.

In canine ileum we investigated the distribution of pituitary adenylate cyclase-activating peptide (PACAP), using immunofluorescence and radioimmunoassay and the binding of 125I-PACAP-27 to membranes. Nerve profiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was present in approximately 100-fold lesser amounts than VIP. High-performance liquid chromatography analysis of deep muscular plexus (DMP) synaptosomes suggested the presence of PACAP-38, PACAP-27, and a third unidentified molecular form. High- and low-affinity 125I-PACAP-27 binding sites were found in DMP synaptosomes and circular smooth muscle (CM) plasma membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants (Kd) and maximal binding capacities (Bmax) were as follows: KdH = 66.9 pM, BmaxH = 101 fmol/mg; KdL = 2.18 nM, BmaxL = 580 fmol/mg protein. The binding of 125I-PACAP-27 was fast. Dissociation was slow and incomplete in the presence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). GTP gamma S or cholera toxin treatment eliminated high-affinity binding in both membranes. VIP had approximately 100-fold lower affinity than PACAP-27 in both membranes. Cross-linking studies identified an approximately 70-kDa PACAP receptor in each membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly Gs-coupled, receptors in DMP synaptosomes and CM membranes.