Organization and development of peptide-containing neurons in the airways.
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Humans↗
Biomedical subjects
R F Coburn
Publications and source records attributed to R F Coburn.
Phorbol ester effects on coupling mechanisms during cholinergic contraction of swine tracheal smooth muscle.
1. We studied effects of the phorbol ester, phorbol 12,13-dibutyrate (PDB), on carbachol-induced contractions of swine trachealis muscle. PDB (1-10 microM) markedly inhibited 5.5 microM-carbachol-induced inositol phosphate synthesis allowing us to study (a) whether the membrane potential-independent component of force (pharmacomechanical coupling component) developed in carbachol-stimulated trachealis muscle is dependent on activation of inositol phospholipid metabolism, and (b) whether carbachol-induced membrane depolarization and contraction are altered in muscle where second messenger signals generated by inositol phospholipid metabolism are inhibited and activation of protein kinase C (PKC) is already maximal. 2. Application of PDB (10 microM) to unstimulated trachealis muscle resulted in a small slowly developing contraction associated with a 10 m V membrane depolarization. PDB-evoked contractions were not influenced by Na+ or Cl- ion substitutions, or administration of amiloride, all of which inhibited PDB-evoked membrane depolarization. 3. Pre-treatment with PDB had no effect on [K+]-force, or [K+]-membrane potential relationships, over a range of extracellular [K+] from 40 to 70 mM. Pretreatment with PDB had no effect on extracellular [Ca2+]-force relationships during 40 mM-K+. 4. Carbachol-evoked contractions of muscle treated with PDB became similar to K+ contractions in regard to effects of organic Ca2+ antagonist drugs or decrease in bathing solution [Ca2+]. At low carbachol concentrations, verapamil plus PDB completely inhibited force development. With 5.5 microM-carbachol, over 90% of total carbachol-induced force was inhibited by verapamil, or nifedipine, plus PDB. 5. Control carbachol-evoked contractions were associated with 20-25 mV membrane depolarizations. In PDB-treated muscle, carbachol-evoked contraction occurred with a blunted depolarization, i.e. about 5 mV. 6. Force controlled by pharmacomechanical coupling mechanisms operating during maintained carbachol-evoked contractions was inhibited by treatment with PDB. Carbachol-induced force dependent on pharmacomechanical coupling mechanisms could be explained by signals generated via inositol phospholipid metabolism. 7. Electromechanical coupling mechanisms were augmented during carbachol in PDB-treated muscle. This appears to be due primarily to changes in the properties or number of surface membrane voltage-gated Ca2+ channels. 8. Data suggest an important role of PKC-mediated phosphorylations for control of both pharmacomechanical coupling mechanisms mediated by activation of inositol phospholipid metabolism and electromechanical coupling mechanisms mediated by effects on operation of surface membrane ion channels.
Amiloride↗
Inositol lipid turnover and compartmentation in canine trachealis smooth muscle.
We established conditions for the study of metabolism and compartmentation of inositol phospholipids in canine trachealis muscle. Unstimulated muscle was incubated with myo-[3H]inositol for 30 min at 37 degrees C which resulted in labeling of the tissue free myo-inositol pool, whereas only a small amount of radioactivity was incorporated into inositol phospholipids or inositol phosphates. After addition of 5.5 microM carbachol, phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2), specific radioactivities increased exponentially, reaching apparent constant values in 180-240 min. Initial rates of increases in PI, PIP, and PIP2 specific radioactivities were 39, 32, and 66 times that measured in unstimulated muscle. Metabolic flux rates (nmol.100 nmol total lipid Pi-1.min-1) during development of force averaged 0.42 +/- 0.09 and during force maintenance averaged 0.14 +/- 0.01. Fractions of total PI, PIP, and PIP2 pools that were linked to muscarinic cholinergic activation were estimated to be 0.97, 0.85, and 0.65, respectively. Initial rates of increase in specific radioactivities and specific radioactivities during carbachol activation were similar in PI, PIP, and PIP2 fast active compartments, suggesting metabolic flux from PI to PIP to PIP2 was in near chemical equilibrium. Turnover times for PI, PIP, and PIP2 fast active compartments were estimated to be 21, 1.6, and 4.0 min, respectively.
Animals↗
Phosphocreatine and oxidative metabolism-contraction coupling in rabbit aorta.
Oxidative metabolism-contraction coupling was studied in single rabbit aortic rings by measuring changes in phosphocreatine (PCr) and ATP content, the ratio of PCr to free creatine (Cr), and changes in muscle force under conditions in which cellular energy production was inhibited. We compared rings with markedly different total PCr content by using rings loaded with creatine, control (unloaded) rings, and rings exposed for 30 min to zero-glucose solution. Creatine loading resulted in increases in PCr content to 300% of control. Maintained muscle force generated by 30 microM norepinephrine was decreased by hypoxia, 0.05 and 0.1 mM cyanide, removal of glucose from the bathing media, or glucose removal plus hypoxia. There was no difference between creatine-loaded and unloaded rings in the mechanical effects of these perturbations. Changes in muscle force induced by inhibition of energy production using the various perturbations correlated with changes in PCr/Cr. Data are consistent with oxidative metabolism-contraction coupling being regulated by energy delivery to a reaction or reactions controlling muscle force. Metabolism-contraction coupling is not controlled by total energy stores or by PCr or Cr concentration.
Animals↗
Oxidative metabolism-contraction coupling in smooth muscle.
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Animals↗
Phosphoinositide metabolism and metabolism-contraction coupling in rabbit aorta.
We tested a hypothesis that metabolism-contraction coupling in vascular smooth muscle is controlled by the rate of delivery of energy to ATP-dependent reactions in the inositol phospholipid transduction system that generate second messengers exerting control on smooth muscle force. Rabbit aorta was contracted by norepinephrine (NOR) under conditions of normoxia and hypoxia (bath PO2 less than 40 mmHg), and changes in inositol phospholipid pool sizes and metabolic flux rates (JF) were determined. JF was determined by labeling free cytosolic myo-inositol by incubation of unstimulated muscle with myo-[3H]inositol and then measuring rates of incorporation of this isotope into inositol phospholipids and inositol phosphates when the muscle was activated by NOR. JF measured during maintenance of NOR-induced force was markedly inhibited during hypoxia to 40-50% of that determined during normoxia; rates of increases in inositol phosphate radioactivities were similarly depressed during NOR activation under hypoxia. The hypoxia-induced decrease in JF was associated with four- to fivefold increase in phosphatidylinositol 4-phosphate (PIP) total pool size, suggesting PIP kinase was inhibited and rate limiting. Total pool sizes of phosphatidylinositol, phosphatidylinositol bisphosphate, and phosphatidic acid were unchanged from values seen during activation under normoxia. These data suggest that activation of inositol phospholipid metabolism, which generates inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, is blunted under conditions where aerobic energy production is inhibited. Data are consistent with "rate-limiting" effects of decreased ATP delivery, or decreased phosphate potential, on PIP kinase and reactions that control resynthesis of phosphatidylinositol.
Animals↗
Effects of elevation of phosphocreatine on force and metabolism in rabbit aorta.
Rings of rabbit thoracic aorta were incubated in 40 mM creatine; both intracellular free creatine (Cr) and phosphocreatine (PCr) content increased 300% in 4 h, whereas tissue ATP content and PCr-to-Cr ratio remained constant. Exogenous Cr was taken up and metabolized by creatine kinase; the newly formed PCr was available for tissue metabolism. Compared with control tissue, muscle rings with markedly increased Cr and PCr content showed no difference in either the rate of contraction with norepinephrine or the amplitude of contraction. ATP production rates, computed from mitochondrial and glycolytic metabolism during rest and during maintained contractions, were similar for creatine-loaded and control rings. Under conditions where muscle was developing force, creatine-loaded rings showed a threefold larger decrease in PCr content than untreated rings but no difference in the decrease in PCr/Cr. A similar pattern of PCr depletion was seen when creatine-loaded or untreated NE-contracted muscle was exposed to hypoxia plus zero-glucose medium. In addition, loaded rings showed smaller rates of efflux of lactate than did unloaded rings when exposed to hypoxia in normal glucose medium.
Adenosine Triphosphate↗
Stretch-induced membrane depolarization in ferret trachealis smooth muscle cells.
We determined the effects of increasing the length of the ferret trachealis muscle on smooth muscle membrane potentials recorded on successive impalements by microelectrodes. The preparation included the paratracheal ganglion nerve plexus as well as trachealis muscle. With sustained increases in muscle length over the range 0.5-0.8 to 1.2 maximal length (Lmax), depolarization occurred, which was related to the amplitude of the length increase. Membrane depolarizations were also evoked after stretching to lengths approximately 1.1 Lmax and returning to the control length. Stretch-induced membrane depolarizations developed after the stretch maneuver was complete; were slowly reversible; were not influenced by tetrodotoxin or atropine; were related to stretch rather than to maintained increase in muscle length; were not transmitted to adjacent nonstretched segments of the trachea; and were often associated with slow waves which appear to be secondary to membrane depolarization rather than stretch per se.
Animals↗
Morphological features of spiking and nonspiking cells in the paratracheal ganglion of the ferret.
The present series of experiments was designed to study details of the morphology and connectivity of functionally identified cells located in the paratracheal ganglia of the ferret. The morphology of 11 spiking (AH cells) and seven nonspiking (type B cells) ganglion cells was examined. Intra-axonally injected horseradish peroxidase (HRP) was used as the label. Each spiking and nonspiking cell was identified by intracellular recording prior to the HRP injection. "Whole mount preparations" were processed for HRP histochemistry with diaminobenzidine as the chromogen. HRP-labeled cell bodies of both the spiking AH and nonspiking type B neurons demonstrated similar morphological features. Both types of ganglion cells showed axons arising from a small, ill-defined axon hillock which exited from the cell as single or multiple branches of equal diameter and coursed unidirectionally through the interganglionic nerve trunk to an adjacent ganglion; short, fine, tapering processes (presumptive dendrites) in the immediate vicinity of the injected cell; and processes extending out of the ganglion cell perpendicular to the interganglionic nerve trunk which could be followed into the smooth muscle. Extraperikaryal injections of HRP into a ganglion retrogradely labeled perikarya in the adjacent ganglia. These results demonstrate that in airway ganglia the morphology of spiking and nonspiking neurons is remarkably similar despite electrophysiological differences. In addition it appears that ganglion cells project to adjacent ganglia and to smooth muscle by means of independent axonal processes. These morphological features of the ganglion cells in airways and the trajectories of their axons correspond to known features of their physiology: i.e., the axon of a ganglion cell travels unidirectionally toward the adjacent ganglion and arborizes there, providing anatomical evidence of communication between ganglia via the interganglionic nerve trunk; and the spiking and nonspiking neurons possess similar morphological features that are typical of ganglion cells described in other systems, such as in the myenteric plexus.
Action Potentials↗
Oxygen-dependent tension in vascular smooth muscle. Does the endothelium play a role?
We investigated a hypothesis that an oxygen sensor involved in hypoxia-induced relaxation of vascular smooth muscle may reside in endothelial cells. We also determined the oxygen dependence of hypoxia-induced decreases in cyclic guanosine 3',5'-monophosphate concentrations in vascular smooth muscle rings. Rings of canine femoral artery, rabbit thoracic aorta, and lamb ductus arteriosus, either with an intact endothelium or with damaged or absent endothelium, were studied using organ baths that allowed changes in PO2 without a change in pH. Hypoxia-induced relaxations of rabbit thoracic aorta, lamb ductus arteriosus, and canine femoral artery were not dependent on an intact endothelium. The magnitude of hypoxia-induced relaxations was unchanged in rings of canine femoral artery without intact endothelium compared to rings with endothelium. Quasi-steady state organ bath PO2-mechanical tension relationships were unchanged in rings of canine femoral artery without endothelium over an organ PO2 range of 200-20 mm Hg. With rabbit thoracic aorta, magnitudes of hypoxia-induced relaxations were significantly smaller in rings without endothelium. Quasi-steady state plots, where mechanical tension was given as percentage of maximal relaxation, were similar in rings either with or without intact endothelium. Cyclic guanosine 3',5'-monophosphate concentrations were shown to be oxygen-sensitive, decreasing during hypoxia-induced relaxations with a threshold PO2 of 80-100 mm Hg with canine femoral artery, and 60-80 mm Hg with rabbit thoracic aorta rings, but this finding seems unrelated to the mechanism of hypoxia-induced relaxation.
Animals↗
Phosphoinositide hydrolysis and smooth muscle function.
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Animals↗
Pharmacomechanical coupling in smooth muscle may involve phosphatidylinositol metabolism.
Cholinergic contraction of canine trachealis muscle, a contraction that primarily utilizes membrane potential-independent mechanisms for activating contractile proteins (pharmacomechanical coupling), is associated with a decline in the phosphatidylinositol pool, an increase in the phosphatidic acid and diacylglycerol pools, and an increased incorporation of 32PO4 into phosphatidylinositol. We found that these changes occur during development of the contraction and during maintenance of tension and are independent of membrane depolarization or increases in cytosolic Ca2+ concentration. These findings suggest that phosphatidylinositol turnover may be part of a receptor transduction process controlling receptor-operated Ca2+ channels or other membrane potential-independent mechanisms involved in pharmacomechanical coupling in smooth muscle.
Animals↗
Electrical and anatomic characteristics of cells of ferret paratracheal ganglion.
A preparation has been developed for study of the electrical properties of the neurons of the ferret paratracheal ganglia. Two cell types were identified. AH cells were characterized by the presence of a single action potential in response to cathodal current pulses, followed by a profound after hyperpolarization that lasted for several hundred milliseconds. Electrical stimulation of branches of the laryngeal nerves produced a fast excitatory postsynaptic potential (EPSP) that was often followed by an apparent inhibitory postsynaptic potential (IPSP). A similar response was seen with electrical stimulation of interganglionic nerve trunks. Hexamethonium reversibly inhibited fast EPSPs in these cells. Type B cells could not be excited by intracellular current injections. At least some of these cells appear to be neurons as electrical stimulation of nerve trunks resulted in a slow EPSP and, rarely, a fast IPSP. Ganglia have between 10 and 20 cell bodies, and their diameters are between 15 and 40 microns. Two types of nerve endings were seen: 1) those with mainly round agranular vesicles 50-60 nm in diameter, and 2) those with large dense-cored vesicles of approximately 100 nm with an electron-lucent halo around the core.
Animals↗
Neural coordination of excitation of ferret trachealis muscle.
The distribution of junction potentials and synaptic potentials to muscle cells and ganglion cells in the ferret trachealis muscle-nerve plexus preparation was studied with local electrical stimulation of branches of the laryngeal nerve or the interganglionic nerve trunk. Stimulations evoked excitatory junction potentials in muscle cells and fast excitatory postsynaptic potentials in ganglion (AH) cells located throughout the preparation, regardless of the location of the stimulating electrode. Evoked excitatory junction potentials were nearly simultaneous in widely separated muscle cells, suggesting that excitation of different muscle cells is coordinated. The apparent conduction velocity to muscle cells after a local nerve stimulation was 0.1-0.2 m/s. Dissemination of input to ganglion cells and muscle cells was dependent on the integrity of the interganglionic nerve trunk. There was evidence based on analysis of conduction velocities that coupling of electrical activity in different AH ganglion cells and muscle cells was related to the following: 1) an interlacing arrangement of myelinated preganglionic neurons that enter the plexus from multiple branches of the laryngeal nerve, and 2) interlacing neural circuits characterized by synapses between neurons whose cell bodies lie in different ganglia.
Animals↗
The anatomy of the ferret paratracheal parasympathetic nerve-ganglion plexus.
Silver-impregnated preparations of the ferret paratracheal nerve-ganglion plexus were studied for description and quantitation of the general morphology of ganglia and nerve bundles and trunks. Most ganglia were found in one or two chains, separated by interganglionic nerve trunks, running the length of the trachea in close apposition to the trachealis muscle. Ganglia were flat, one cell deep, and characterized by irregular spacing and small and variable numbers of perikarya. Perifascicular, infrafascicular, and mixed types of ganglia were seen. The mean perikaryon density was 127 +/- 20/cm2 of trachealis muscle. In addition to connections to the interganglionic nerve trunk, more than 70% of ganglia were also apparently "connected" to nerve bundles running to the periphery and/or the center of the preparation, and the plexus seemed considerably more complex than originally described. There was no significant change in density, size or type, or connections of ganglia with nerve bundles in the cervical or thoracic trachea.
Animals↗
Effect of incubation of guinea-pig taenia coli in potassium-free media on arachidonate release and lipid metabolism.
We studied the effects of immersion of guinea-pig taenia coli strips in potassium-free media on arachidonate stores and other lipid fractions. Control studies obtained with the strips in Krebs solution showed that greater than 97% of arachidonate was found esterified in phospholipid with the following distribution: phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol. 30 min incubation of the strips with [3H]arachidonate complexed to albumin resulted in incorporation of this isotope into phospholipid and neutral lipid fractions, phosphatidylcholine greater than neutral lipid greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. 30 min incubations with 32PO4(2-)-resulted in an isotope incorporation into phospholipids, phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. After 'loading' with [3H]arachidonate and 32P, placing the strips in potassium-free media caused the following: there was an increased release of [3H]arachidonate from the tissue into the bathing solution. [3H]Arachidonate and 32P radioactivity in phosphatidylinositol fell without a change in phosphatidylinositol content. [3H]Arachidonate and 32P radioactivity in other phospholipid fractions was unchanged. Arachidonate specific activity fell and arachidonate content increased in the phosphatidylserine plus phosphatidylinositol fraction. [3]Arachidonate in neutral lipid did not change significantly. We conclude that exposure of taenia coli to potassium-free media activates turnover of phosphatidylinositol, which results in release of arachidonate.
Animals↗
Inhibitory innervation to the guinea pig trachealis muscle.
The inhibitory innervation of the cervical trachea was studied in situ in anesthetized male guinea pigs. We measured effects of electrical stimulation of vagal motor and sympathetic trunk nerve fibers, during atropine, on trachealis muscle tension. Effects of direct transmural stimulation of trachealis muscle were also determined. We confirmed the dual nature of the inhibitory innervation to this muscle. Vagal motor inhibitory nerves are shown to be preganglionic. Neural transmission at the level of the ganglia is characterized by filtering of high frequency action potentials. The neurotransmitter at the myoneural junction is unidentified but is not norepinephrine. Maximal relaxation accounts for about 20-40% of maximal relaxations seen with transmural stimulation of trachealis muscle in the presence of atropine. Sympathetic trunk nerve fibers are also preganglionic. Neurotransmission at the level of the ganglia is apparently 1:1 at high-action potential frequencies. Norepinephrine released presynaptically has access to smooth muscle beta- but not alpha-receptors. Maximal adrenergic relaxations account for 60-80% of total transmural stimulation relaxations. Transmural stimulation relaxations appear to be accounted for by release of neurotransmitter from sympathetic adrenergic plus vagal nonadrenergic postganglionic nerve fibers.
Adrenergic Fibers↗