Inhibition of norepinephrine-induced contractile responses of canine mesenteric artery by plant total saponins.
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Animals↗
Biomedical subjects
E E Daniel
Publications and source records attributed to E E Daniel.
Peptide-containing nerves around blood vessels of stroke-prone spontaneously hypertensive rats.
The distribution and density of nerves containing vasoactive intestinal polypeptide, substance P, and neuropeptide Y around the cerebral and peripheral blood vessels of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied using an indirect immunofluorescence technique. Neonatal sympathectomy of SHRSP with anti-nerve growth factor and guanethidine was also carried out to study the effect of sympathectomy on the distribution of these nerves. Vasoactive intestinal polypeptide nerve density was higher in the veins and superior mesenteric artery of SHRSP than of WKY and lower in the cerebral arteries of SHRSP than of WKY, but no difference was found in the muscular mesenteric arteries. Sympathectomy reduced the density of these nerves in all the peripheral vessels but had little effect on the cerebral arteries. Density of substance P nerves was similar between SHRSP and WKY in the peripheral vessels but higher in the cerebral arteries of WKY than of SHRSP. Sympathectomy reduced the density of these nerves in the peripheral vessels but increased the density in some cerebral arteries of SHRSP. Neuropeptide Y nerve density was higher in the peripheral blood vessels of SHRSP than of WKY, and no difference was found in the cerebral arteries. Sympathectomy almost completely removed these nerves in the peripheral vessels but had no effect on the cerebral arteries. We suggest that some of the differences in nerve density between SHRSP and WKY, especially those in the peripheral blood vessels, may be related to the development of hypertension in the SHRSP.
Animals↗
Elevations in arterial pressure induce the formation of spontaneous action potentials and alter neurotransmission in canine ileum arteries.
Arterial segments (less than 250 micron o.d.) excised from canine ileum were mounted in a chamber that permitted arterial transmural pressure (TMP) to be altered and measured. Subsequently, the periarterial nerves were field stimulated with single pulses (0.1 msec, 70 V), and the resting membrane potential (Em) as well as the nerve-mediated alterations in smooth muscle Em were measured using intracellular microelectrodes at TMPs between 0 and 160 mm Hg. The resting Em was greatest at TMPs of 40 mm Hg (-54.7 +/- 2.6 mV) and depolarized as the TMP was increased, reaching a value of -44.8 +/- 3.1 mV at 160 mm Hg. At TMP greater than or equal to 60 mm Hg, a proportion of the preparations exhibited spontaneous electrical activity (SA) consisting of constant rhythmic oscillations in Em or action potentials (APs) or of trains of rhythmic APs that progressively decreased in amplitude, interrupted by periods of hyperpolarization. SA stopped when the TMP was lowered to 40 mm Hg and was reestablished when the TMP was reelevated to TMPs above 60 mm Hg. Nerve stimulation evoked excitatory junction potentials (ejps) or APs. At constant stimulus parameters, ejps of maximum amplitude having the greatest rate of potential rise and fall were produced at TMP of 100 mm Hg. At TMPs greater than 100 mm Hg or less than 100 mm Hg, the amplitude and the rate of rise and fall of the ejps decreased. Ejps formed in response to a constant single pulse stimulus (0.1 msec, 70 V) elicited APs only at TMPs greater than or equal to 60 mm Hg. Neither ejps nor APs were inhibited by alpha-receptor-blocking agents. These studies indicate that the TMP at which an artery is maintained plays an important role in determining the resting Em, the occurrence of spontaneous action potentials, and the alterations in Em associated with nerve stimulation.
Action Potentials↗
Receptors for tachykinins in canine intestine circular muscle.
125I-Tyr8-substance P binding was examined in a plasma membrane enriched fraction from the circular muscle of canine small intestine. The binding was quick in onset, reversible and saturable to a single high affinity binding site; KD and maximum receptor concentration were 0.50 = 0.06 nM and 742 +/- 173 fmol/mg of protein, respectively. KD values from kinetic and saturation studies were in agreement. The rank order of potency of the tachykinins and related compounds in displacing this binding was consistent with that of a neurokinin (NK)-P (NK-1) type binding site. 125I-eledoisin did not bind specifically to these membranes and eledoisin was relatively ineffective in displacing the 125I-Tyr8 substance P. Kassinin was totally ineffective. Based on relative agonist potencies, the tachykinins appeared to contract the circular muscle of canine small intestine in vitro by a mechanism which also could be considered to be a NK-P (NK-1) type receptor. However, a comparison of data obtained by these two techniques suggests that the NK-P (NK-1) type binding site and the receptor(s) responsible for contractile responses are not identical. There were discrepancies in relative orders of potency and in absolute potencies. The simplest explanation is that there is also an NK-A (NK-2) receptor which is functional but not labeled by our ligands. Possible reasons for these discrepancies are discussed.
Animals↗
Effects of inflammatory mediators on neuromuscular transmission in canine trachea in vitro.
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15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Neural transmission during ozone-induced airway hyperresponsiveness.
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Acetylcholine↗
Alpha-2 adrenoceptors on nerves and muscles of rat uterus.
To test the hypothesis that functional postsynaptic alpha-2 adrenoceptors exist in rat myometrium, we examined whether specific binding sites for [3H]rauwolscine were present on microsomal membranes from myometrium of nonpregnant, day 16 pregnant and delivering rats and whether an alpha-2 adrenoceptor agonist has functional effects. The myometrium of rats at term undergoes a physiological denervation, confirmed in this study by ultrastructural examination of uterine samples and by [3H]saxitoxin binding studies. Binding sites for rauwolscine of similar Kd (11-15 nM) were present in all groups of myometrium and were localized on plasma membranes. There was no significant change in the density of rauwolscine binding sites in membranes from day 16 animals compared to nonpregnant ones, but a significant fall (38%) from these values at term. Strips of uterine circular or longitudinal muscle from nonpregnant, day-16 or day-22 pregnant rats failed to respond to the selective alpha-2 agonist, BHT-920, in the presence of propranolol; i.e., BHT-920 neither caused contraction nor inhibited contractions induced by oxytocin. BHT-920 did not affect the inhibitory effects of isoproterenol which were antagonized by propranolol. However, it antagonized contractions to norepinephrine in the presence of propranolol with a pKB value of 5.6 to 5.7. These contractions were phentolamine-sensitive. BHT-920 displaced rauwolscine from binding to all groups of myometria (IC50 = 2 to 3 x 10(-6) M) and displaced prazosin (Kd = 0.65 nM) from binding to myometria of nonpregnant rats (IC50 value congruent to 2 x 10(-4) M). Phentolamine also displaced rauwolscine from binding (IC50 = 2 x 10(-8) M). 5-Hydroxytryptamine displaced rauwolscine from binding only at higher concentrations (IC50 greater than 10(-5) M). We conclude that binding sites for alpha-2 adrenoceptor antagonists in myometrial microsomes were located primarily on smooth muscle plasma membrane. A smooth muscle alpha-2 adrenoceptor agonist appeared to occupy a site on muscle with the same affinity as it displayed toward rauwolscine binding site and competitively inhibited effects of an alpha-1 agonist. Our data suggest that alpha-2 adrenoceptor binding sites may exist on smooth muscle without coupling to contractile function, but their occupancy competitively prevents occupancy of alpha-1 agonist receptor activation sites.
5'-Nucleotidase↗
Local actions of trimebutine maleate in canine small intestine.
A study of the local actions of trimebutine (TMB) maleate and its N-diesmethyl metabolite (TMB-M) was carried out in the gastrointestinal tract of anesthetized dogs. In the unstimulated small intestine, but not in the stomach or colon, i.a. TMB and TMB-M caused activation of circular muscle. Like the activation by i.a. [Met5]-enkephalin, this was antagonized by naloxone. In field-stimulated segments of stomach and small intestine circular muscle, TMB or TMB-M, like dynorphin-1-13 or [Met5]-enkephalin, inhibited the phasic and tonic contractions which were mediated mostly by cholinergic, postganglionic nerves. However, the inhibitory effects of dynorphin-1-13 or [Met5]-enkephalin on small intestine were antagonized by naloxone whereas those of TMB sometimes or those of TMB-M usually were not. TMB or TMB-M did not affect responses to i.a. acetylcholine, but high doses reduced the contractile responses to subsequent field stimulation and excitatory responses to [Met5]-enkephalin. We concluded that the excitatory local actions of TMB or TMB-M on small intestine involved opioid receptors probably of the mu or delta types. Inhibitory local actions on nerve-mediated responses, however, may not have involved opioid receptors. Comparison of these data to results when TMB or TMB-M were given i.v. suggests that these agents also have peripheral actions to affect gastrointestinal motility at sites outside the gastrointestinal tract.
Animals↗
Ultrastructure of airway smooth muscle.
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Animals↗
Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin.
We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.
Animals↗
Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding.
To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.
Animals↗
The projections of chemically identified nerve fibres in canine ileum.
The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.
Animals↗
Ultrastructural localization of VIP-immunoreactivity in canine distal oesophagus.
The localization of vasoactive intestinal polypeptide (VIP) immunoreactivity in canine distal oesophagus was studied using different fixation and embedding procedures and labelling with protein A-gold. The retention of immunoreactivity and the preservation of ultrastructure was best after fixation with a mixture of 0.1% glutaraldehyde and 4% paraformaldehyde, and embedding in 'LR White' resin using the 'cold-cured' method. VIP immunoreactivity was localized exclusively over large granular vesicles in the myenteric plexus and in nerves of the circular muscle. Most varicose profiles in circular muscle were labelled, but some large granular vesicles in the same profiles which contained labelled vesicles as well as some varicose profiles with large granular vesicles were unlabelled. From these data it was uncertain whether unlabelled large granular vesicles contained VIP or other neuropeptides. A striking finding was the dense and close innervation of the interstitial cells of Cajal with nerve containing VIP-labelled large granular vesicles. This finding is consistent with earlier suggestions that VIP-immunoreactive nerves may innervate these cells which are in gap junction contact with smooth muscle and that this arrangement may be involved in the transmission of non-adrenergic, non-cholinergic nerve activity in distal oesophagus.
Animals↗
Elastin and elastase-like enzyme change in aorta of rat with malignant hypertension.
In order to study the role of elastin in arteries with respect to hypertension and hypertensive arterial disease, aortic elastin content and elastase-like enzyme activity were examined and compared in stroke-prone spontaneously hypertensive rats (SHRSP), which show malignant hypertension, and Wistar-Kyoto normotensive rats (WKY). The elastin content was lower, whereas the elastase-like activity was higher at 20 weeks of age in SHRSP than in WKY, so that the aortic elastin/enzyme ratio of SHRSP was lower than that in WKY. These differences were not found at 6 weeks of age (prehypertensive stage). For SHRSP anti-hypertensive treatment resulted in lowering the elastase-like activity and in increasing the elastin content in comparison to untreated animals. The subcellular distribution of the elastase-like activity closely correlated with that of 5'-nucleotidase activity, a plasma membrane marker enzyme. The results indicate involvement of a smooth muscle plasmalemmal elastase-like enzyme in vascular connective tissue metabolism in health and possibly also its participation in hypertensive arterial diseases.
Aging↗
Subcellular fractionation of the longitudinal smooth muscle/myenteric plexus of dog ileum: dissociation of the distribution of two plasma membrane marker enzymes.
The distribution of plasma membrane markers, the sodium pump [evaluated as ouabain-sensitive, potassium-stimulated p-nitrophenyl phosphatase (K+-pNPPase)], [3H]saxitoxin binding, and 5'-AMPase, was studied in the subcellular fractions prepared from the homogenates of the longitudinal smooth muscle/myenteric plexus of dog ileum. The K+-pNPPase activity and [3H]-saxitoxin binding were found to be predominantly associated with the synaptosomal fraction as indicated by the high level of these activities in the crude synaptosomal fraction and by the copurification of K+-pNPPase and [3H]saxitoxin binding, but not 5'-AMPase, with several synaptosomal markers during the fractionation of the crude synaptosomal fraction on density gradients. In contrast to the K+-pNPPase activity and [3H]saxitoxin binding, the 5'-AMPase activity was found to be concentrated in the microsomal pellet. Further fractionation of microsomes on density gradient resulted in copurification of 5'-AMPase but not K+-pNPPase or [3H]saxitoxin binding, with other smooth muscle plasma membrane-bound enzymes, such as high-affinity Ca2+-ATPase, Mg2+-ATPase, and Ca2+-ATPase. It was concluded that in the longitudinal smooth muscle/myenteric plexus, the sodium pump activity is present in higher density in the neuronal plasma membranes whereas 5'-AMPase activity is concentrated in the smooth muscle plasma membranes.
4-Nitrophenylphosphatase↗
Catecholamines release mediators in the opossum oesophageal circular smooth muscle.
1. Effects of catecholamines applied exogenously to the circular smooth muscle layer of the body of the oesophagus of the opossum (Didelphis marsupialis) were studied, simultaneously measuring changes in the membrane potential, the membrane conductance and the contractility of the muscle, using the double sucrose-gap technique. 2. Superfusion of the smooth muscle with Krebs solution at 27 degrees C containing dopamine (10(-6)-10(-4) M) dose-dependently caused a hyperpolarization of the smooth muscle cells and an increased membrane resistance followed after gradual repolarization by oscillations of the membrane potential, often accompanied by muscle action potentials. During the hyperpolarization, the tendency for the membrane potential to sag during prolonged application of hyperpolarizing currents was reduced and the 'off' depolarization following such currents was increased. This muscle did not develop active tension prior to treatment; it therefore did not relax during the hyperpolarizations, but contracted following the depolarized phase of oscillations. 3. The non-adrenergic, non-cholinergic nerve-mediated inhibitory junction potential (i.j.p.) showed a small reduction in amplitude during superfusion with dopamine, explicable as a result of the drug-induced hyperpolarization. The 'off' response following the i.j.p., decreased transiently when the membrane potential was hyperpolarized to its maximum value. Then it increased to values larger than control as the membrane repolarized. Vasoactive intestinal polypeptide (VIP, 10(-6) M) produced a similar response but hyperpolarizations were smaller. 4. Of the tested catecholamines, isoprenaline, phenylephrine, butylated hydroxytoluene-920 (BHT-920) and clonidine were ineffective whereas the potency order for other catecholamines was dopamine greater than noradrenaline greater than or equal to adrenaline greater than DOPA. The catecholamine-induced responses were not affected by alpha- or beta-adrenoreceptor antagonists given alone or in combination. Of the dopamine receptor antagonists tested domperidone was without effect, whereas haloperidol reduced and bulbocapnine blocked the response. The findings suggested that a receptor resembling DA1-type peripheral receptor mediated the effects of dopamine on opossum oesophagus. 5. The catecholamine-induced responses and those to VIP disappeared completely in Cl-(-)free medium (isethionate replacement). 6. Conditioning depolarization of the smooth muscle cells decreased but hyperpolarization increased the amplitude of the hyperpolarization (up to 20 mV). With larger hyperpolarizations the responses decreased and disappeared at around 50 mV hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Effects of endogenous and exogenous prostaglandin in neurotransmission in canine trachea.
The effect of PGE2 on neurotransmission in the canine tracheal strip dissected free of epithelium was studied in the single sucrose gap and organ bath. PGE2 was a potent inhibitor of the initiation of excitatory junction potentials (ejps) by just submaximal nerve stimulation. In a concentration of 10(-9) or 10(-8) M PGE2 nearly or completely abolished them. Contractile responses to field stimulation in the sucrose gap at 27 degrees C or in muscle baths at 37 degrees C were also reduced or abolished by PGE2 in the same dose range; reductions were greater at low frequency. Responses to acetylcholine were also depressed but significantly less than to field stimulation. These are consistent with major presynaptic as well as some postsynaptic inhibitory actions of PGE2. No evidence was obtained that endogenous PGE2 affected excitatory junction potentials and contractions; i.e. they were stable for hours and unaffected by indomethacin 10(-6) and 10(-5) M under our conditions. Post-stimulus potentiation of ejps amplitude, maximum at 10 s, was observed and became more marked after the first ejp had been markedly reduced or abolished by PGE2. This potentiation was unaffected by indomethacin. It was suggested that a presynaptic process inhibited by PGE2 might participate in this potentiation. The canine trachea is a useful preparation when studied under the experimental condition used here for study of effects of products of arachidonate on neurotransmission.
Acetylcholine↗
Muscarinic receptors on nerves and muscles in opossum esophagus muscularis mucosa.
The muscarinic receptors of muscularis mucosa have some recognition properties that suggest they resemble receptors of the M1 subtype. The nerves of these tissues also contain muscarinic receptors which inhibit tonic contractions caused by release of a substance-P-like material by field stimulation. These receptors also appear to be M1 in type as they are maximally activated by McNeil A343 as well as by carbachol (pD2, 5.5 and 7.5, respectively). They are also inhibited by pirenzepine, as well as by atropine (negative logarithms of the required dose for 50% inhibition or potentiation, 6.6-6.7 compared with 8.2-8.3). Hexahydrosiladifenidol, an antagonist selective or M2 receptors of guinea pig ileum, had a low (approximately 7.1) pA2 value for antagonism of both agonists in smooth muscle in this tissue. However, it was closer to atropine in potency with respect to potentiating tonic responses to field stimulation or to inhibiting phasic responses to field stimulation than it was to antagonizing smooth muscle contractions. Thus, atropine was about 40 times more potent than pirenzepine and 2-5 times more potent than hexahydrosilafenidol. There were some quantitative differences in the effectiveness of these three antagonists in blocking the phasic (acetylcholine-mediated) response to field stimulation. Atropine was 70-100 times more potent than pirenzepine and 8-25 times more potent than hexahydrosiladifenidol. This greater potency difference for inhibition of phasic contractions compared with potentiation of tonic contractions was discussed. This tissue appears to be one of the first smooth muscles in which both nerves and muscles contain muscarinic receptors with some recognition properties resembling those of the M1 subtype.
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗