Pituitary adenylate cyclase-activating peptide as a neurotransmitter in the canine ileal circular muscle.

Pituitary adenylate cyclase-activating peptide (PACAP)1-27, PACAP1-38, and vasoactive intestinal peptide (VIP) initiated dose-dependent contractions of canine ileal circular muscle after intra-arterial injection in vivo or ex vivo. PACAP1-27- and VIP-induced contractions approached the tissue maximum; VIP was 100-fold less potent. PACAP1-38 was more potent than VIP. PACAP1-27 contractions in vivo were unaffected by hexamethonium, reduced equally by atropine or atropine plus hexamethonium, and abolished by tetrodotoxin (TTX), suggesting that PACAP released acetylcholine and another excitatory neurotransmitter from postganglionic cholinergic enteric nerves. In myenteric plexus-free circular muscle strips, PACAP1-27 at 10(-9) M and PACAP1-38 or VIP at 10(-7) M increased [3H]acetylcholine release during nerve stimulation, suggesting the locus of one postganglionic site at which PACAP1-27 acts. All agonists inhibited nerve-mediated contractions in vivo with a potency rank order similar to that for excitation. Inhibition of nitric oxide (NO) synthetase or TTX decreased the duration and amplitude of PACAP1-27- but not PACAP1-38-induced inhibition. Inhibition of NO synthetase abolished VIP-induced inhibition, but TTX did not. Submaximal contractions to acetylcholine were amplified by PACAP1-27 or VIP before TTX and inhibited after TTX. Thus, both PACAP molecules and VIP directly inhibit and indirectly excite smooth muscle contractions. PACAP1-27 and VIP also release NO. The functional potency differences between PACAP1-27 and VIP suggest PAC1 receptors mediate all responses, likely through the stimulation of adenylate cyclase.

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.

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.

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.

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.

Nitric oxide (NO) inhibits release of acetylcholine from nerves of isolated circular muscle of the canine ileum: relationship to motility and release of nitric oxide.

A method was developed to study simultaneously 1) release of ACh from nerve varicosities and terminals in and 2) motility of the myenteric plexus-free circular muscle of the canine ileum. Tissues were incubated with 3H-choline. Release of 3H was analyzed before and during electrical field stimulation of nerves in the presence and absence of choline oxidase. The major components released into the superfusate during field stimulation were (in the absence of choline oxidase) 3H-ACh and 3H-choline as determined by column chromatography and (in the presence of choline oxidase) the choline metabolite 3H-betaine as isolated by tetraphenylboronbutyronitrile extraction. Addition of tetrodotoxin (1 x 10(-6)M) or superfusion with Ca(++)-free Krebs reduced motor activity and the release of 3H-ACh, which confirmed that release was mediated from nerve varicosities. Addition of Nw-nitro L-arginine methyl ester or Nw-nitro L-arginine increased field-stimulated 3H-ACh release. This effect was reduced in the presence of L-arginine but not D-arginine. Motility was also increased by the addition of Nw-nitro-L-arginine methyl ester and Nw-nitro L-arginine. In contrast, the addition of L-arginine did not restore motor activity to control levels, and D-arginine had no effect. Our findings that inhibition of nitric oxide synthesis amplifies ACh release during field stimulation suggest that field stimulation releases both ACh and nitric oxide from nerve terminals of the deep muscular plexus and that this nitric oxide inhibits field-stimulated ACh release and circular muscle contractility.

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.

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.

Peptide YY stimulates circular muscle contractions of the isolated perfused canine ileum by inhibiting nitric oxide release and enhancing acetylcholine release.

Peptide YY (PYY) and neuropeptide Y (NPY), infused into the quiescent isolated perfused canine ileum, dose-dependently increased phasic activity of the circular muscle and decreased tonic output of immunoreactive vasoactive intestinal peptide (VIP) in the venous effluent. The contractions subsided before the prolonged inhibition of VIP output. Motor excitation by PYY, an abundant neuropeptide in this tissue, was reduced by blockade of muscarinic or nicotinic receptors, or inhibition of nitric oxide (NO) synthase, despite continued inhibition of VIP output. A combination of atropine and hexamethonium eliminated the PYY-induced decrement in VIP output and left motor excitation unchanged. Blockade of NO synthase eliminated motility increases under these conditions. Intracellular microelectrode recordings of myenteric plexus-free circular muscle strips found no effect of NPY on the resting membrane potential, or on the field stimulation-induced inhibitory junction potential. Inhibition of VIP release plays no essential role in changing motility. These results suggest that PYY/NPY induce motility by stimulating release of acetylcholine and inhibiting NO release at a locus proximal to but not on nerve terminals.

Galanin inhibition of vasoactive intestinal polypeptide release and circular muscle motility in the isolated perfused canine ileum.

The role of porcine galanin, infused arterially into isolated perfused canine ileal segments, in modulating the tonically elevated neural release of vasoactive intestinal polypeptide and possible concomitant motor actions dependent on vasoactive intestinal polypeptide modulation was studied. Galanin infusions (9-minute) inhibited vasoactive intestinal polypeptide release in a concentration-dependent manner (maximum during minutes 8-10) irrespective of the absence (quiescence) or presence of phasic circular muscle contractions induced by local electrical field stimulation of nerves. During quiescence, galanin induced phasic contractions in four of five segments beginning in the 8th minute of the infusion. During stimulated contractions, galanin inhibited phasic motor activity within 2 minutes of initiation of the infusion; this inhibition may result from direct smooth previously reported muscle inhibition. Thus galanin may inhibit both neural release of vasoactive intestinal polypeptide and circular muscle motility directly. The delayed period of phasic activity initiated by galanin during quiescence may be related to inhibition of vasoactive intestinal polypeptide release, freeing the muscle from tonic inhibition by vasoactive intestinal polypeptide. Because galanin and vasoactive intestinal polypeptide are colocalized in some enteric nerves, galanin may regulate vasoactive intestinal polypeptide release by negative feedback.

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.

[Effect of work of different types and intensity on changes in work capacity]. / Wplyw pracy o róznym charakterze i intensywnosci na zmiany zdolnosci wysilkowej.

Changes in the current effort capacity following 4-hrs' performance of six types of work of different intensity and different muscular system engagement, have been evaluated. The studies involved two groups of women. Group I, consisting of 25 women, performed the dynamic, work on cycle-and manual-ergometers (Rr) and two types of static effort resulting from the shift of the load constituting 20% body weight (N) and maintenance of forced posture during manual weaving (T). Group II, consisting of 9 women, performed the dynamic work on cycle ergometer (R) and dynamic-static work, with an additional load, i.e. holding--in one's hand--2,5 kg (R2,5) and 5.0 kg (R5,0). The effort capacity was determined before and after work, basing on an ergometer test. Whatever the type of the previously performed work, the tolerance of fatigue was found to decrease, which was expressed by lower values of oxygen consumption and heart rate at which the women stopped the test effort. The intensity of the previously performed work (% VO2 max) might be estimated from the difference in the average test effort power, respiratory quotient value and amount of oxygen consumed per one power unit. The static load was evidenced by changes in the blood systolic pressure and increased heart rate during the effort test.