NANC Relaxation Research Articles

O-226 - L-NAME-sensitive and -insensitive NANC relaxation of cat airway in vivo and in vitro studies

O-226 - L-NAME-sensitive and -insensitive NANC relaxation of cat airway in vivo and in vitro studies

Opioids modulate the cholinergic contraction but not the nonadrenergic relaxation in guinea-pig airways in vitro.

(D-ALa2, NMePhe4, Gly-ol5) encephalin (DAMGO), a selective mu-opioid receptor agonist, has previously been demonstrated to inhibit the cholinergic and the noncholinergic contraction in guinea-pig airways. In contrast, opioids had no inhibitory effect on cholinergic neurotransmission in the upper trachea when stimulated at 8 Hz. We investigated whether DAMGO, a selective mu-opioid receptor agonist, [D-Pen 2,5] encephalin (DPDPE), a selective delta-opioid receptor agonist, and U-69593, a selective kappa-opioid receptor agonist could modulate the cholinergic contraction in the upper trachea at different frequencies of stimulation. Moreover, we have investigated whether DAMGO, DPDPE and U-69593 could also modulate the iNANC relaxation. DAMGO (1-100 microM) inhibited the cholinergic contraction in the upper trachea with a maximum inhibition of 57+/-15% at 1 Hz (n=4; p<0.05). On the other hand, DPDPE (10 microM) and U69593 (10 microM) did not produce any significant inhibition of the cholinergic contraction. Naloxone, an opioid receptor antagonist (100 microM), was able to antagonize the inhibitory effect of DAMGO (n=5; p<0.01) on the cholinergic contraction at a frequency of 2 Hz. DAMGO (10 microM) did not displace the cumulative concentration-response relationship to acetylcholine (10 nM-10 mM), (n=4; NS). This provides evidence that prejunctional mu-opioid receptors (and not delta-opioid or kappa-opioid receptors) modulate cholinergic contraction in the upper trachea. In contrast, DAMGO (10 microM) had no significant inhibitory effect on the nonadrenergic relaxation (n=4; NS) in the upper trachea. Neither DPDPE nor U-69593 had any effect on the nonadrenergic relaxation. These findings suggest that DAMGO directly inhibits the cholinergic contraction and that the opioid receptor involved in the inhibition of the cholinergic contraction in the upper trachea is of the mu-opioid type. The finding that opioids inhibit cholinergic contraction without altering NANC relaxations suggests that distinct populations of nerves mediate these two effects.

Non-adrenergic non-cholinergic nerve-mediated inhibitory control of pigeon oesophageal muscle.

Pigeon oesophageal smooth muscle in vitro has spontaneous electromechanical activity. In the presence of atropine and guanethidine, electrical field stimulation evokes a transient TTX-sensitive response comprising inhibition of electric bursting activity and muscular relaxation. This NANC inhibitory response was analysed using the K+ channel blockers TEA and apamin, TEA perfusion (0.1-5 mM) induced a concentration-dependent reduction in amplitude of EFS-evoked relaxation. Responses to higher stimulation frequencies were more sensitive to TEA than those to lower ones. The maximum reduction in amplitude (29% of control) was obtained on 30 Hz EFS evoked responses during 5 mM TEA perfusion. In a similar way, apamin (0.01-10 microM) perfusion reduced NANC relaxation, up to 30% of control. These results suggest that in the pigeon oesophagus, NANC intramural neurons are responsible for muscular relaxation. We speculate that an increase in K+ conductance might be the main mechanism involved, although the residual response after K+ channel blockade indicates the existence of an additional ionic mechanism.

P-589 - Involvement of protein kinases in NANC relaxation of longitudinal muscle of rat distal colon.

P-589 - Involvement of protein kinases in NANC relaxation of longitudinal muscle of rat distal colon.

P-563 - Regional difference in the distribution of L-NAME sensitive and -insensitive NANC relaxation in feline airway.

P-563 - Regional difference in the distribution of L-NAME sensitive and -insensitive NANC relaxation in feline airway.

Regional difference in the distribution of L‐NAME‐sensitive and ‐insensitive NANC relaxations in cat airway.

1. To investigate the distribution profile of functional inhibitory non-adrenergic non-cholinergic (i-NANC) nerves and the contribution of NO to the NANC relaxation in the cat, we studied the effects of N omega-nitro-L-arginine methyl ester (L-NAME) on NANC relaxation elicited by electrical field stimulation (EFS) in the trachea, bronchus and bronchiole. 2. EFS applied to the tracheal smooth muscle during contraction induced by 5-HT (10(-5) M) in the presence of atropine (10(-6) M) and guanethidine (10(-6) M) elicited a monophasic NANC relaxation. By contrast, NANC relaxation elicited in the peripheral airway was biphasic, comprising an initial fast followed by a second slow component and L-NAME (10(-5) M) selectively abolished the first component without affecting the second one. In the trachea, L-NAME (10(-5) M) completely suppressed the monophasic NANC relaxation when single or short repetitive stimuli (< 5) with 1 ms pulse duration were applied. However, at higher repetitive stimuli (> 10) with 1 or 4 ms pulse duration, suppression of NANC relaxation was incomplete. 3. In the small bronchi obtained from L-NAME-pretreated cats, EFS applied during contraction induced by 5-HT (10(-5) M) elicited only the slow component of NANC relaxation which is sensitive to tetrodotoxin. 4. In the peripheral airway, a newly synthesized VIP antagonist (10(-6) M) or alpha-chymotrypsin (1 U ml-1) considerably attenuated the amplitude of L-NAME-insensitive relaxation. 5. Single or repetitive EFS consistently evoked excitatory junction potentials (EJPs) in the central and peripheral airways. When tissues were exposed to atropine (10(-6) M) and guanethidine (10(-6) M), single or repetitive EFS did not alter the resting membrane potential. 6. These results indicate that at least two neurotransmitters, possibly NO or NO-containing compounds and VIP, are involved in i-NANC neurotransmission and the distribution profile of the two components differs in the central and peripheral airway of the cat.

FORCE AND INTRACELLULAR Ca2+ DURING NANC‐MEDIATED RELAXATION OF RAT ANOCOCCYGEUS MUSCLE AND THE EFFECTS OF CYCLOPIAZONIC ACID

1. Simultaneous recordings of tension and [Ca2+]i during NANC-mediated relaxation were made in the rat anococcygeus muscle under various conditions. 2. In muscles precontracted with guanethidine, nitrergic stimulations at 2 Hz produced a rapid decrease in both the tension and [Ca2+]i. 3. The nitric oxide synthase inhibitor, NG-nitro-L-Arginine (NOLA, 100 mumol/L) completely abolished the decreases in the [Ca2+]i and force response of the NANC-mediated relaxation. 4. Noradrenergic-mediated contractions elicited by electrical field stimulation were potentiated by the addition of NOLA. In the absence of NOLA, the motor responses were larger in magnitude at 10 Hz stimulation than at 2 Hz. After NOLA, both the force response and the associated rise in [Ca2+]i were substantially increased in comparison to the control stimulations. Proportionately the potentiation of the 2 Hz response was of a far greater magnitude than that of the 10 Hz response. 5. The guanylate cyclase inhibitor methylene blue (10 mumol/L), partially inhibited the force and [Ca2+]i response of the NANC relaxation. 6. Following exposure of the muscles to the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, cyclopiazonic acid, (10 mumol/L) the responses to NANC stimulation were inhibited. The attenuated relaxation response displayed a bi-phasic timecourse and the Ca2+ change in comparison to that of the control was markedly smaller. In some cases, a relaxation was observed with no detectable change in the [Ca2+]i. 7. The results suggest that part of the relaxation response observed with NANC-mediated relaxation in the rat anococcygeus is dependent on Ca2+ sequestration into the sarcoplasmic reticulum. However, other Ca2+ lowering mechanisms and possible Ca2+ independent mechanisms may also contribute to the NANC relaxation response.

Maturation of nonadrenergic noncholinergic inhibitory system in normal and allergen-sensitized rabbits.

We investigated the functional existence of the nonadrenergic noncholinergic inhibitory (NANCi) system in developing rabbit airways in vitro. Furthermore, we evaluated the effect of parenteral exposure to a specific allergen (ragweed) on the maturation of this neural pathway. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from normal and ragweed-sensitized New Zealand White rabbits at 1, 2, 4, and 12 wk of age. The TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit solution, atropine (1 x 10(-5) M), and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation was applied at stimulation frequencies ranging from 5 to 30 Hz to determine the frequency that produced maximal relaxation. The NANCi response to EFS was measured and expressed as the mean (+/- SE) percent relaxation at 20 Hz, because this stimulation frequency gave the maximal NANCi response at each age studied. TSM segments obtained from control rabbits at 1 wk of age did not demonstrate a NANCi response at the frequencies of stimulation used. By contrast, a reproducible NANC relaxation was demonstrated in TSM from 2-, 4-, and 12-wk-old rabbits. The magnitude of this response was 27 +/- 4.7 (n = 10), 29 +/- 4.8 (n = 9), and 37 +/- 4% (n = 18), respectively. The same experiments performed on TSM segments obtained from ragweed-sensitized animals gave significantly decreased values of NANCi response. In 2-, 4-, and 12-wk-old rabbits, the NANCi responses were 11.5 +/- 3.4 (n = 9), 11 +/- 2 (n = 13), and 16 +/- 4.2% (n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

NANC neurotransmission in the bovine retractor penis muscle is blocked by superoxide anion following inhibition of superoxide dismutase with diethyldithiocarbamate

This study examined the effects of inhibiting Cu/Zn Superoxide dismutase with diethyldithiocarbamate (DETCA) on the ability of Superoxide generating agents such as pyrogallol, hypoxanthine/xanthine oxidase and LY 83583, to influence NANC relaxation of strips of bovine retractor penis (BRP) muscle. Although pyrogallol (100 μM) and hypoxanthine (0.3 mM)/xanthine oxidase (64 mU ml −1) had little effect on NANC relaxation in control strips, both induced almost complete inhibition following treatment with DETCA (3 mM) for 1 h. Inhibition was due to the actions of Superoxide anion since it was blocked by the addition of exogenous Superoxide dismutase (250 U ml −1). LY 83583 (0.1–30 μM) produced a concentration-dependent inhibition of NANC relaxation even in control strips and this too was blocked by exogenous Superoxide dismutase, but sensitivity to inhibition was enhanced 10-fold following treatment with DETCA. The data suggest that under normal circumstances the NANC neurotransmitter is protected by high levels of tissue Superoxide dismutase, and inhibition of this enzyme increases its susceptibility to destruction by Superoxide anions. An important impediment to accepting free nitric oxide as the NANC neurotransmitter in the BRP on the basis that Superoxide anion-generating agents inhibit the actions of authentic nitric oxide but not those of NANC nerve stimulation has thus been removed.

Inhibitory nerve distribution and mediation of NANC relaxation by nitric oxide in horse airways.

The distribution of inhibitory nerves and the mediator of the inhibitory nonadrenergic noncholinergic (iN-ANC) nervous system were investigated in smooth muscle preparations from seven regions of equine airways. In tissues incubated with atropine and precontracted with histamine, electrical field stimulation produced frequency-dependent relaxation, and the magnitude of the relaxation decreased from trachea to central bronchi and was absent in peripheral airways. The degree of relaxation in bronchi was not simply a function of bronchial size or generation. Propranolol inhibited part of the relaxation only in the cranial trachealis. After propranolol, NG-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, eliminated the remaining relaxation in all preparations. This effect was reversed by L-arginine, the NO precursor, but not by D-arginine. Exogenous NO concentration dependently relaxed trachealis. These results indicate that: 1) adrenergic innervation is limited to cranial trachealis, 2) iNANC nerves supply the trachea and central bronchi, and 3) NO mediates iNANC function.

NADPH-diaphorase and NANC relaxations are correlated in the sheep urinary tract

The present investigation was performed with the purpose of revealing by histochemical examination of NADPH-diaphorase activity and electrical field stimulation (EFS) of isolated preparations in vitro, whether a nitrergic innervation is present in the lower urinary tract of the sheep. NADPH-diaphorase positive fibers were found in the trigone and urethra, but not in detrusor and ureter. EFS elicited l-nitroarginine-sensitive relaxations of precontracted preparations from the trigone and urethra while it did not relax detrusor and ureteral preparations. The present results show a direct regional correlation between the NADPH-diaphorase activity and EFS-induced relaxations, and suggest the presence of an inhibitory nitrergic innervation, which might be of importance for relaxation of the bladder neck and urethra during voiding.

O-67 - Actions of nitrosocystine and L-NG-nitro-arginine (L-NNA) on NANC relaxation, resting membrane potential and EJPs of cat tracheal smooth muscle cells.

O-67 - Actions of nitrosocystine and L-NG-nitro-arginine (L-NNA) on NANC relaxation, resting membrane potential and EJPs of cat tracheal smooth muscle cells.

The effect of L-NG-nitro arginine on non-adrenergic, non-cholinergic (NANC) relaxations in the longitudinal muscle of the guinea pig proximal colon

We have investigated the effect of the NO synthesis inhibitor L-NG-nitro arginine (L-NNA) on the NANC relaxation induced by electrical field stimulation (FS) or the 5-HT1-like receptor agonist 5-carboxamidotryptamine (5-CT) in the longitudinal muscle of the guinea pig proximal colon. In the presence of atropine (0.2 microM) and guanethidine (5.0 microM), NANC relaxations induced by FS (0.1-10 Hz, 160 mA, 0.3 ms) or 5-CT (1.0-30 microM) were completely abolished by tetrodotoxin (0.3 microM). L-NNA (10-100 microM) produced concentration-dependent inhibition of NANC relaxations, but D-NNA had no effect. This inhibitory effect of L-NNA on NANC relaxations was prevented by L-arginine (1 mM) or L-citrulline (1 mM), but was not affected by D-arginine (1 mM). Like L-NNA, L-NMMA (100-300 microM) also inhibited the NANC relaxation induced by FS and 5-CT. These results suggest that nitric oxide (NO) or a substance releasing NO may contribute to NANC relaxations of guinea pig proximal colon induced by FS and the stimulation of 5-HT1-like receptors.