Eicosanoids and peripheral neurotransmission.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
E E Daniel
Publications and source records attributed to E E Daniel.
Vasoactive intestinal polypeptide and non-adrenergic, non-cholinergic inhibition in lower oesophageal sphincter of opossum.
1. Field stimulation or vasoactive intestinal polypeptide (VIP) relaxed lower oesophageal sphincter (LOS) from North American opossum. Pretreatment with carbachol in Cl-ion-containing or Cl-ion-free Krebs solution or with 10(-3) M 9-aminoacridine abolished or markedly reduced relaxation due to VIP applied exogenously but not that elicited by field stimulation of non-adrenergic, non-cholinergic nerves. 2. Inhibitory junction potentials (7.5 +/- 1.2 mV, n = 5) could be recorded in LOS strips with the sucrose gap technique. They lacked significant after-depolarizations but were accompanied by decreased membrane resistance (61 +/- 6%, n = 3). In these strips, VIP (10(-6) M) produced small hyperpolarizations (2.1 +/- 1.1 mV, n = 5) sometimes followed by membrane potential oscillations but no change in conductance. 3. Removal of external chloride depolarized the membranes (7.6 +/- 1.7 mV) but did not prevent the hyperpolarization to VIP or the occurrence of inhibitory junction potentials. Restoration of external chloride repolarized the cells. It appears that an appreciable chloride conductance may be present in sphincter muscle cells and this may cause them to be more depolarized than non-sphincter muscle. 4. We conclude that it is very unlikely that VIP is the inhibitory NANC neurotransmitter since it does not mimic the inhibitory junction potential.
Animals↗
Classification of tachykinin receptors in muscularis mucosae of opossum oesophagus.
1. Muscularis mucosae of the distal oesophagus of the opossum contracts in response to substance P and to a variety of tachykinins. To delineate the nature of the receptors present in this tissue, we evaluated contractile responses to substance P, neurokinin A, neurokinin B, eledoisin and analogues believed to be highly selective for NK-1, NK-2 and NK-3 receptors. In addition, the effects of prolonged exposure to each of these agents (10(-6) M or 10(-5) M) on contractile responses to substance P and to itself were evaluated. Similarly effects of prolonged exposure to the various tachykinins and their analogues on the field-stimulated responses of this muscle were studied. 2. All naturally occurring tachykinins were full agonists and differed in potency (comparing ED50 values) by less than ten fold. In nearly all cases there was cross tachyphylaxis between substance P and the other tachykinins and each reduced tonic responses to field stimulation, a response previously shown to be mediated by a substance P like agent. Eledoisin failed to cause tachyphylaxis under the conditions of these experiments. 3. When highly selective tachykinin analogues were used, only that believed to activate NK-1 receptors was a full agonist. [beta-Ala4,Sar9,MetO2(11)]SP(4-11) was also only slightly less potent than substance P. In contrast, an agonist selective for NK-2 (NK-A) receptors, [Nle10]NKA(4-10), and one selective for NK-3 (NK-B) receptors, [beta-Asp4, MePhe7]NKB(4-10) were unable to produce a response equal to 50% of the maximum even at 10(-5) M. However, all three selective tachykinin analogues reduced responses to substance P but not to carbachol. They usually reduced both phasic and tonic responses to field stimulation. 4. We conclude, based on this and earlier study, that the tachykinin receptors of opossum oesophagus muscularis mucosae recognize all naturally occurring tachykinins but may represent only NK-1 receptors. The ability of analogues selective for other types of tachykinin receptors to reduce responses to substance P raises the possibility that their selectivity depends in part on diminished efficacy rather than totally on diminished affinity at some classes of receptor.
Animals↗
Characterization of alpha-adrenoceptor subtypes by [3H]prazosin and [3H]rauwolscine binding to canine venous smooth muscle membranes.
Postsynaptic alpha-adrenoceptor subtypes were studied using [3H]prazosin and [3H]rauwolscine binding to plasmalemma-enriched microsomal fractions isolated from dog saphenous veins and mesenteric veins. Both radioligands showed saturable binding consistent with the presence of a single homogeneous binding site in each case, based on Scatchard analysis. The Kd values of [3H]prazosin and [3H]rauwolscine, calculated from kinetic studies were similar to those from equilibrium binding data in both venous muscle membranes. The microsomal membranes of dog saphenous vein and mesenteric vein contained about a fourfold higher density of the high affinity [3H]rauwolscine binding sites than those for [3H]prazosin binding. In competition studies, IC50 values for displacement of rauwolscine or prazosin suggested that the sites of interaction for the antagonists prazosin and rauwolscine were independent. Phenylephrine, a functionally selective alpha-adrenoceptor agonist, competed with a similar IC50 value for the specific binding sites of [3H]prazosin and [3H]rauwolscine; but B-HT 920, a functionally selective alpha 2-adrenoceptor agonist, competed for [3H]rauwolscine and [3H]prazosin binding with distinctly different IC50 values. Our data show the existence of two populations of alpha-adrenoceptor antagonist binding sites in the plasma membranes of dog saphenous vein and mesenteric vein, and raise the question whether agonist selectively depends on different affinities or on differential efficacies at one or two sites.
Animals↗
Functional studies of nerve projections in the canine intestine.
An in vivo model has been developed to study nerve connections in the canine intestine, using spread of field stimulated contractions recorded proximally and distally with strain gauges and local intra-arterial injections of drugs. Excitation spread orally for several centimetres, more effectively at lower frequencies of field stimulation. This excitation was blocked by local hexamethonium or by a combination of atropine and naloxone (each of which reduced the contractions). Distal excitation occurred after a longer delay than oral excitation; during the delay there was frequently an initial relaxation response. Distal excitation was greater at higher frequencies of field stimulation, but like oral excitation it was blocked by hexamethonium or by a combination of atropine and naloxone. Distal relaxation responses were unaffected by atropine or naloxone, but were abolished by hexamethonium. "Off" contractions, those that followed cessation of field stimulation, occurred at higher frequencies of field stimulation proximally and distally near the site of field stimulation and were blocked by atropine but not by naloxone or hexamethonium. The effects of all agents given locally extended beyond the sites of injection. These results suggest that a chain of cholinergic nerves with nicotinic synapses transmit excitation orally and distally to circular muscle; these effects seem to be facilitated proximally and distally by opioid nerves and to be inhibited initially distally by a noncholinergic mechanism. Explanations of these findings are proposed.
Animals↗
Does in vitro activation of postsynaptic alpha 2-adrenoceptor utilize intracellular Ca2+ for contraction in dog saphenous vein?
Dog saphenous vein spiral strips were employed to determine whether an intracellular source of Ca2+ is used for contraction upon activation of the alpha 2-adrenoceptor by B-HT 920 in Ca2+-free Krebs solution containing 50 microM EGTA. The studies were carried out in parallel with the activation of the alpha 1-adrenoceptor by phenylephrine (Phe) under the condition that B-HT 920 (10(-5) M) and Phe (2 x 10(-6) M) gave rise to a similar level of responses in Ca2+-containing Krebs solution. A similar level of responses to these agonists at equieffective concentrations in Ca2+-free medium were also observed. Such responses to Phe and B-HT 920 were inhibited by 10(-7) M rauwolscine and 10(-7) M prazosin, respectively, and were not affected by 10(-7) M nifedipine or 5 mM Mn2+. The responses to B-HT 920 (10(-5) M) and submaximal concentration of Phe (2 x 10(-6) M) in Ca2+-free medium were additive. However, if the vascular strips were first contracted maximally with 10(-4) M Phe in Ca2+-free medium to deplete the intracellular Ca store, subsequent addition of B-HT 920 failed to induce additional response. Our results strongly suggest that activation of alpha 2-adrenoceptor in dog saphenous vein in Ca2+-free medium indeed utilizes intracellular Ca2+ for contraction. We also found that the failure of earlier studies to demonstrate the contractile effects of B-HT 920 in dog saphenous vein was due to experimental artifacts derived from the use of high concentration of EGTA and artificial pH-buffering reagent.
Animals↗
Morphology of the canine pyloric sphincter in relation to function.
The ultrastructure and immunocytochemistry of the canine distal pyloric muscle loop, the pyloric sphincter, were studied. Cells in this muscle were connected by gap junctions, fewer than in the antrum or corpus. The sphincter had a dense innervation and a sparse population of interstitial cells of Cajal. Most such cells were of the circular muscle type but a few were of the type in the myenteric plexus. Nerves were sometimes associated with interstitial cell profiles, but most nerves were neither close to nor associated with interstitial cells nor close to smooth muscle cells. Nerve profiles were characterized by an unusually high proportion of varicosities with a majority or a high proportion of large granular vesicles. Many of these were shown to contain material immunoreactive for vasoactive intestinal polypeptide (VIP) and some had substance P (SP) immunoreactive material. All were presumed to be peptidergic. VIP was present in a higher concentration in this muscle than in adjacent antral or duodenal circular muscle. Interstitial cells of Cajal made gap junctions to smooth muscle and to one another and might provide myogenic pacemaking activity for this muscle, but there was no evidence of a close or special relationship between nerves with VIP or SP and these cells. The absence of close relationships between nerves and either interstitial cells or smooth muscle cells leaves unanswered questions about the structural basis for previous observations of discrete excitatory responses or pyloric sphincter to single stimuli or nerves up to one per second. In conclusion, the structural observations suggest that this muscle has special neural and myogenic control systems and that interstitial cells may function to control myogenic activity of this muscle but not to mediate neural signals.
Animals↗
Improved propagation in myometrium associated with gap junctions during parturition.
The hypothesis that gap junction (GJ) formation between myometrial cells at term improves electrical coupling was tested. We measured the spread of electrical excitation from six extracellular electrodes aligned on uterine strips in either the longitudinal (axial) or transverse (circumferential) direction. Spontaneous bursts propagated over the entire 15-mm recording distance in the axial direction at both preterm and parturition and showed some characteristics of a system of coupled relaxation oscillators. However, individual spikes within the bursts propagated further and with higher velocity at parturition than at preterm. In the circumferential direction, both bursts and individual spikes propagated further at parturition than before. Propagation in this axis at parturition appeared to require an intact circular muscle layer. Spikes evoked by electrical stimulation also propagated further and with higher velocity in both axes at parturition. Electron microscopy showed many GJs between uterine smooth muscle cells during parturition, but few and sometimes no GJs at preterm. Thus improved propagation was associated with increased GJ contact between myometrial cells, consistent with the hypothesis that gap junction formation at term improves electrical coupling.
Action Potentials↗
Pacemaker activity recorded in interstitial cells of Cajal of the gastrointestinal tract.
The hypothesis was tested that interstitial cells of Cajal can generate slow wave activity. Intracellular recordings were performed only in the most superficial cells at the submucosal surface of the canine colonic circular muscle layer. An omnipresent and characteristic slow wave activity was present in all cells with a mean amplitude of 37 +/- 3 mV, a frequency of 4.6 +/- 0.1 counts/min (cpm), and a duration of 5.6 +/- 0.5 s; the average resting membrane potential was -70 +/- 1 mV. To determine the type of cell from which these recordings were obtained, methylene blue was injected by microiontophoresis. The strips were immediately fixed while the microelectrode was kept in the cell. A small segment of the tissue containing this cell was then processed for electron microscopy and serially sectioned. Electron-microscopic evidence showed that the microelectrode tip was positioned in an interstitial cell of Cajal (ICC): 1) several sections were observed with round cytoplasmic lesions of decreasing diameter followed by sections from the same cell without the lesion and 2) electron-dense material was observed in these sections due to the injected methylene blue. These cells were identified as part of the ICC network present at the muscle-submucosa interface of the circular muscle and were positively identified as ICC by the presence of cell processes. This is the first report giving direct evidence for the occurrence of electrical slow waves in ICC. It is essential support for the hypothesis that ICC are the actual pacemaker cells of the gut musculature.
Animals↗
Inhibitory function of VIP-PHI and galanin in canine pylorus.
Contractions of canine pylorus and adjacent muscles were recorded with antral and duodenal strain gauges and a Dent sleeve manometric assembly with additional side holes in the pylorus. In vivo, vasoactive intestinal polypeptide (VIP) and peptide histamine isoleucine (PHI) given through the gastroepiploic artery inhibited both spontaneous and acetylcholine-induced pyloric contractions, before and after neural blockade with tetrodotoxin. Galanin inhibited only nerve-stimulated contractions. VIP greater than PHI much greater than galanin inhibited pyloric motor activity initiated by field stimulation of duodenal intramural nerves or intraduodenal infusion of 0.1 N HCl. In vitro, forskolin or field stimulation of nerves inhibited contractions of the pyloric muscle induced by various agonists but VIP-PHI failed to inhibit these contractions on two-thirds of the strips. The in vivo results suggest that VIP (or PHI) may be an inhibitory mediator in the pylorus acting directly on smooth muscle. Galanin may inhibit neural excitatory pathways in the pylorus. However, the structures on which VIP and PHI act in vivo may have become nonfunctional in vitro and other mediators account for the neural inhibition observed in isolated strips.
Acetylcholine↗
Distribution and function of enteric GAL-IR nerves in dogs: comparison with VIP.
The distribution of nerves containing galanin-immunoreactive (GAL-IR) material was compared to the distribution of neurons containing vasoactive intestinal polypeptide (VIP) immunoreactivity in the canine gastrointestinal tract. The actions of intra-arterially administered galanin and VIP on motility in the gastric antrum and corpus and the intestines were also studied. All sphincter muscles contained galanin- and VIP-immunoreactive nerve profiles. VIP-immunoreactive nerve profiles were present in all layers of the stomach, small intestine, and colon. GAL-IR nerve somata were common in the submucous plexus of ileum and colon and in the myenteric plexus of the terminal antrum, as were nerve processes in various layers. In the small intestine, galanin inhibited contractile responses to field stimulation of intrinsic nerves and also reduced the contractions after nerve blockade with tetrodotoxin (TTX). VIP often enhanced field-stimulated contractions at low doses but inhibited these and the contractions after TTX at higher doses. In the stomach and colon, both peptides inhibited responses to field stimulation; whether these effects were due to actions on smooth muscle was not tested. The distribution and actions of galanin in gut are consistent with the hypothesis that it acts at smooth muscle sites and possibly at prejunctional sites.
Animals↗
Biochemical studies on opioid and alpha 2-adrenergic receptors in canine submucosal neurons.
Biochemical information about receptors for adrenergic and opioid neurotransmission in submucosal plexus (SMP) is unavailable. We have purified a fraction P2 enriched in synaptosomes and neuronal membranes (high [3H]saxitoxin binding and high vasoactive intestinal polypeptide immunoreactivity, low activity of 5'-nucleotidase) from the canine small intestine SMP. The synaptosomal fraction (fraction P2) also contained a high density of opioid diprenorphine binding sites of high affinity. [3H]rauwolscine binding was enriched both in fraction P2 and in a microsomal fraction. Competition experiments using several adrenergic and opioid receptor ligands revealed that opioid receptors were approximately 64% mu-, 24% delta-, and 12% kappa-subtypes and that adrenoceptors on fraction P2 were alpha 2-subtype but that there was a heterogeneous population of alpha 2-adrenoceptors. These studies show that a fraction enriched in synaptosomes and neural membranes from the canine intestine SMP contains opioid as well as alpha 2-adrenoceptors, that all three subtypes of opioid receptors seem to be present with mu-receptors predominant, and that subtypes of alpha 2-adrenoceptors appear to be present.
5'-Nucleotidase↗
Distribution of opioid receptors in canine small intestine: implications for function.
Distribution of the binding sites for [3H]diprenorphine, a non-selective opiate ligand, was studied in membrane fractions from longitudinal muscle/myenteric plexus and circular muscle containing deep muscular plexus. [3H]saxitoxin was used as a marker for neuronal plasma membranes and 5'-nucleotidase as a marker for smooth muscle plasma membranes. Saxitoxin binding correlated strongly with diprenorphine binding, but 5'-nucleotidase correlated poorly with diprenorphine or saxitoxin binding in these fractions. Opiate binding sites in membranes of myenteric and deep muscular plexus were of high affinity (Kd = 0.12 and 0.18 nM, respectively) with maximum binding capacity of 400 and 500 fmol/mg protein, respectively. Competition experiments using subtype-selective opiate ligands indicated that all three subtypes of opiate receptors were present in the same ratio of 40-45% mu-subtypes, 40-45% delta-subtypes, and 10-15% kappa-subtypes on both plexuses. Opiate receptors of canine small intestine, therefore, are located primarily or exclusively on nerves with similar distributions in nerve membranes containing only axonal varicosities (deep muscular plexus) as in those containing neurons, dendrites, and varicosities (myenteric plexus).
Animals↗
Release of VIP and substance P from isolated perfused canine ileum.
Basal release of vasoactive intestinal polypeptide (VIP) was 100-fold higher than substance P (SP) release from the vascularly perfused, neurally isolated canine small intestine. High-frequency field stimulation increased SP release but decreased VIP release. VIP release was markedly reduced by tetrodotoxin, perfusion with Ca-free medium, or by hexamethonium but not by atropine. Acetylcholine increased VIP output by an atropine-sensitive mechanism. Methionine-enkephalin or dynorphin (at 10-fold higher concentrations) markedly reduced VIP output; the actions of both these were abolished by naloxone. BHT-920, an alpha 2-adrenoceptor agonist, reduced VIP output markedly by a rauwolscine-sensitive mechanism. Isoproterenol and phenylephrine were without effect. Motilin produced persistent inhibition of VIP output. Thus VIP neurons of isolated canine small intestine were continuously active, driven by intrinsic cholinergic nerves, and subject to presynaptic inhibition by opioid agonists, alpha 2-adrenoceptor agonists, and motilin. This intrinsic neural system may provide tonic inhibitory control to the small intestinal circular muscle and provide a mechanism by which agents may modulate intestinal motor function.
Animals↗
Improvement in airway responsiveness and asthma severity during pregnancy. A prospective study.
In the myometrium and gut, smooth muscle becomes less contractile during pregnancy, probably because of the effect of progesterone and estrogen. It is not known whether these hormones cause similar changes in airway smooth muscle, and therefore, this study examined airway responsiveness during the large hormonal changes of pregnancy and evaluated whether changes in responsiveness are associated with changes in (1) progesterone and estrogen and (2) the clinical severity of asthma. Twenty nonpregnant asthmatic women were assessed every 3 months until conception. In the 16 who conceived, assessments were repeated once during the second and third trimesters and 1 month after delivery. Data collected preconception and from those who did not conceive within 1 yr were used for control subjects. There was a 2-fold improvement in airway responsiveness during pregnancy from a preconception mean PC20 0.35 to 0.72 mg/ml during the second trimester and 0.58 mg/ml during the third (p = 0.03). Post-delivery responsiveness (0.48 mg/ml) was not significantly different from preconception. The improvements during pregnancy were greater than the 3 monthly fluctuations when not pregnant (p = 0.04) and were greatest in those who were most hyperresponsive initially (p = 0.01). There was an associated improvement in clinical asthma severity as indicated by a reduction in minimum medication requirements (p = 0.03) and this was not at the expense of good symptoms control; both symptoms and spirometry remained unchanged during pregnancy. Changes in responsiveness were not closely related to progesterone or estriol, suggesting that other nonhormonal factors may also contribute to the improvement during pregnancy and that the control of asthmatic airway smooth muscle may not be exactly the same as that of the myometrium and gut.
Adult↗
Epinephrine facilitates neurogenic responses in isolated segments of dog mesenteric arteries.
Epinephrine (EPI) bitartrate (10(-9)-10(-8) M) significantly enhanced tension development in response to electrical field stimulation in isolated segments of dog mesenteric arteries. Responses to exogenous norepinephrine (NE) were generally unaffected, indicating that the EPI-induced increases in response to field stimulation are not explicable in terms of changes in postjunctional sensitivity. The facilitatory effects of EPI (5 X 10(-9) M) were unaffected by atenolol (10(-6) M) but were completely abolished by timolol (2 X 10(-7) M), suggesting an involvement of beta 2-adrenoreceptors in mediating the EPI-induced facilitation of neurogenic responses. Responses to exogenous NE were usually unaffected by either beta-adrenoreceptor antagonist. The results suggest that release of endogenous sympathetic neurotransmitter, measured in terms of postjunctional effects on vascular smooth muscle tone, appears to be modulated by prejunctional facilitatory beta 2-adrenoreceptors and that these receptors may be a physiologic site of action of EPI.
Animals↗
Relation between density (maximum binding) of alpha adrenoceptor binding sites and contractile response in four canine vascular tissues.
In microsomal fractions from dog aorta, saphenous veins, mesenteric arteries and veins, both [3H]prazosin and [3H]rauwolscine displayed monophasic saturation in binding. The Kd for [3H] rauwolscine binding was similar for all these blood vessels, but the maximum number of [3H]rauwolscine binding sites was 3 to 7 times higher in veins compared to arteries. The Kd for [3H] prazosin was higher in saphenous vein than that in the arteries. The maximum number of binding sites for [3H]prazosin was similar, except for that in aorta, which was 3 times greater. Phenylephrine (alpha-1 adrenoceptor selective agonist) or norepinephrine (nonselective adrenoceptor agonist) produced similar maximal responses in all vessels. The alpha-2 adrenoceptor selective agonist, B-HT 920 (2-amino-6-allyl-3,4,7,8-tetrahydro-6H-thiazolo[5,4-d]-azepine)-induced contraction in veins but not in arteries. Prazosin (10(-6) M) inhibited completely the contractions to norepinephrine (3 x 10(-6) M) in mesenteric arteries and to phenylephrine (3 x 10(-6) M) in arteries and veins. Contractile responses of mesenteric artery were unaffected by rauwolscine. Rauwolscine (10(-7) M) caused a greater parallel rightward shift of the concentration-response curve to norepinephrine than did prazosin (10(-7) M) in saphenous veins, and a further rightward shift of responses to norepinephrine after 10(-7) M prazosin in mesenteric vein and saphenous vein and abolished B-HT 920-induced responses at alpha-2 adrenoceptors. The tissues responding to B-HT-920 correspond to those having the highest alpha-2 receptor density as measured by [3H]rauwolscine binding. The density of such sites required for contraction to be initiated in veins was much higher than with alpha-1 adrenoceptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Identification and characterization of presynaptic and postsynaptic beta adrenoreceptors in the longitudinal smooth muscle/myenteric plexus of dog ileum.
The subcellular distribution of [125I]cyanopindolol (125I-CYP) binding in membrane fractions derived from the longitudinal smooth muscle/myenteric plexus (LSM/MP) of dog ileum followed a bimodal pattern with selective enrichments in the purified synaptosomal (S2) fraction and the purified smooth muscle plasma membrane (M2) fraction. The half-maximal saturation of binding sites in both fractions occurred at 10 pM [125I]CYP with maximal binding capacities, in femtomoles per milligram of protein, 70 +/- 10 (S2 fraction) and 110 +/- 25 (M2 fraction). Approximately 80% of binding sites in either membrane fraction exhibited a high affinity for beta-2 selective antagonist ICI 118,551 (Ki = 1.4 +/- 0.4 nM in S2 and 1.8 +/- 0.6 nM in M2; mean +/- S.D. n = 3), whereas remaining 20% of binding sites had a low affinity for this agent (Ki = 2.6 +/- 1.5 microM in S2 and 0.55 +/- 0.2 microM in M2). The beta-1-selective antagonist ICI 89,406 had a high affinity for approximately 20% of binding sites (Ki = 12 +/- 7 nM in S2 and 4 +/- 2 nM in M2) and a low affinity for approximately 80% of binding sites (Ki = 0.34 +/- 0.16 microM in S2 and 0.42 +/- 0.24 microM in M2). The displacement potencies of beta-adrenergic agonists followed the order (-)-isoproterenol greater than salbutamol (-)-epinephrine greater than (-)-norepinephrine in each membrane preparation. The inclusion of 0.1 mM 5'-guanylylimidodiphosphate into assay medium resulted in a decreased affinity of receptors for agonists and increased values of Hill coefficients.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylyl Cyclases↗