Cellular coupling and conducted vasomotor responses.

1. The present brief review examines the concept of spreading vasodilator responses in arteriolar trees, its physiological relevance and possible mechanisms. 2. The most likely mechanisms involve spread of hyperpolarization through tissues in the vessel wall, made possible by electrical coupling between the cells. It is generally agreed that there is coupling between cells within the muscle and endothelial layers, but coupling between the two layers is not always present. 3. The passive electrical properties of arterioles can be modelled, using different techniques depending on the complexity of branching of the arteriolar tree. Comparison of experimental results with the model indicates that hyperpolarization can spread further than expected from passive properties alone, implying that spreading vasodilatation may be an active process.

Simulating the spread of membrane potential changes in arteriolar networks.

OBJECTIVE: Our aim was to simulate the spread of membrane potential changes in microvascular trees and then make the simulation programs accessible to other researchers. We have applied our simulations to demonstrate the implications of electrical coupling between arteriolar smooth muscle and endothelium. METHODS: A two-layered, cable-like model of an arteriole was used, and the assumptions involved in the approach explicitly stated. Several common experimental situations that involve the passive spread of membrane potential changes in microvascular trees were simulated. The calculations were performed using NEURON, a well-established computer simulation program that we have modified for use with vascular trees. RESULTS: Simulated results show that membrane potential changes would probably not spread as far in the endothelium as they would in the smooth muscle of arterioles. Where feed arteries are connected to larger distributing arteries, passive spread alone may not explain the physiologically observed spread of diameter changes. CONCLUSIONS: Simulated results suggest that the morphology of an arteriole, in which the muscle layer is much thicker than the endothelium, favors electrical conduction along smooth muscle rather than the endothelium. However, it seems that passive electrical spread is insufficient to explain the apparent spread of membrane potential changes in experimental situations. Active responses involving voltage-dependent conductances may be involved, and these can also be included in our simulation.

Electrophysiological basis of arteriolar vasomotion in vivo.

We tested the hypothesis that cyclic changes in membrane potential (E(m)) underlie spontaneous vasomotion in cheek pouch arterioles of anesthetized hamsters. Diameter oscillations (approximately 3 min(-1)) were preceded (approximately 3 s) by oscillations in E(m) of smooth muscle cells (SMC) and endothelial cells (EC). Oscillations in E(m) were resolved into six phases: (1) a period (6 +/- 2 s) at the most negative E(m) observed during vasomotion (-46 +/- 2 mV) correlating (r = 0.87, p < 0.01) with time (8 +/- 2 s) at the largest diameter observed during vasomotion (41 +/- 2 microm); (2) a slow depolarization (1.8 +/- 0.2 mV s(-1)) with no diameter change; (3) a fast (9.1 +/- 0.8 mV s(-1)) depolarization (to -28 +/- 2 mV) and constriction; (4) a transient partial repolarization (3-4 mV); (5) a sustained (5 +/- 1 s) depolarization (-28 +/- 2 mV) correlating (r = 0.78, p < 0.01) with time (3 +/- 1 s) at the smallest diameter (27 +/- 2 microm) during vasomotion; (6) a slow repolarization (2.5 +/- 0.2 mV s(-1)) and relaxation. The absolute change in E(m) correlated (r = 0.60, p < 0.01) with the most negative E(m). Sodium nitroprusside or nifedipine caused sustained hyperpolarization and dilation, whereas tetraethylammonium or elevated PO(2) caused sustained depolarization and constriction. We suggest that vasomotion in vivo reflects spontaneous, cyclic changes in E(m) of SMC and EC corresponding with cation fluxes across plasma membranes.

An equation describing spread of membrane potential changes in a short segment of blood vessel.

The spread of membrane potential changes throughout certain cells and tissues plays an important role in their physiology. The attenuation of such changes in any tissue is usually characterized by the cable length constant lambda, which can be determined experimentally if the equations describing membrane potential spread in the tissue are known. Here we derive an equation describing spread of membrane potential changes in a short cable, which is an appropriate model for short segments of blood vessels. This equation is more general than those already published in that the positions of both the current source that gives rise to a potential change, and the point at which the change is measured, can be anywhere along the cable.

Computerised colour: a technique for the assessment of burn scar hypertrophy. A preliminary report.

A technique for the objective measurement of one parameter of burn scar hypertrophy is described. The technique involves capturing a video camera image on a computer and subsequent quantitative analysis of the colour of the scar using a custom-written computer programme. By analysing multiple areas and comparing the identical areas over the course of treatment, it is theoretically possible to measure the progress and compare differing modes of therapy.

Inhibition of vasoconstriction and Ca2+ currents mediated by neuropeptide Y Y2 receptors.

The effects of neuropeptide Y (NPY) and agonists selective for NPY Y1 and Y2 receptors were studied on contraction and Ca2+ currents in arterial smooth muscle. In isolated arterioles from the guinea pig small intestine, small brief constrictions were evoked by depolarising the arteriolar smooth muscle using high K+ solution applied from a micropipette. The constrictions were reduced in amplitude by the Y2-selective agonists PYY(13-36) and N-acetyl[Leu28, Leu31]NPY-(24-36) in concentrations from 20-100 nM. NPY or the Y1 selective agonist [Leu31 Pro34]NPY in concentrations from 50 pM to 100 nM increased the amplitude of the constrictions, with a maximum effect at 10 nM. Smooth muscle cells were isolated from rat small mesenteric arteries, and voltage-activated Ca2+ currents measured by whole cell patch clamping. The peak amplitude of the Ca2+ currents was decreased by N-acetyl[Leu28, Leu31]NPY-(24-36), and by NPY (100 nM). [Leu31, Pro34]NPY either had no effect or slightly increased the Ca2+ currents. We conclude that Y2 receptors on vascular smooth muscle can reduce Ca2+ currents induced by depolarisation, and thus oppose constriction caused by smooth muscle depolarisation.

Computer simulation of the enteric neural circuits mediating an ascending reflex: roles of fast and slow excitatory outputs of sensory neurons.

Recent electrophysiological studies of the properties of intestinal reflexes and the neurons that mediate them indicate that the intrinsic sensory neurons may transmit to second order neurons via either fast (30-50 ms duration) or slow (10-60 s duration) excitatory synaptic potentials or both. Which of these possible modes of transmission is involved in the initiation of motility reflexes has not been determined and it is not clear and what the consequences of the different forms of synaptic transmission would be for the properties of the reflex pathways. In the present study, this question has been addressed by the use off a suite of computer programs, Plexus, which was written to simulate the activity of the neurons of the enteric nervous system during intestinal reflexes. The programs construct a simulated enteric nerve circuit based on anatomical and physiological data about the number, functions and interconnections of neurons involved in the control of motility. The membrane potentials of neurons are calculated individually from physiological data about the reversal potentials and membrane conductances for Na+, K+ and Cl-. Synaptic potentials are simulated by changes in specific conductances based on physiological data. The results of each simulation are monitored by recording the membrane potentials of up to 16 separate defined neurons and by recording the summed activity of whole classes of neurons as a function of time and location in the stimulated network. The present series of experiments simulated the behaviour of a network consisting of 18,898 sensory neurons and 3708 ascending interneurons after 75% of the sensory neurons lying in the anal 10 mm of a 30 mm long segment of small intestine were stimulated once. The results were compared with electrophysiological data recorded from myenteric neurons during ascending reflexes evoked either by distension or mechanical stimulation of the mucosa. When transmission from sensory neurons to ascending interneurons was via fast excitatory synaptic potentials, the latencies and durations of the simulated responses were too brief to match the electrophysiologically recorded responses. When transmission from sensory neurons was via slow excitatory synaptic potentials, the latencies were very similar to those recorded physiologically, but the durations of the stimulated responses were much longer than seen in physiological experiments. The latencies and durations of simulated and physiologically recorded responses matched only when the firing of ascending interneurons was limited to the beginning of a slow excitatory synaptic (in this study by limiting the duration of the decrease in K+ conductance). The simulation provided several physiologically testable predictions, indicating that Plexus is an important tool for the investigation of the properties and behaviour of the enteric nervous system.

Immunohistochemical and electrophysiological characterization of submucous neurons from the guinea-pig small intestine in organ culture.

Immunohistochemical and electrophysiological properties of submucous neurons were investigated in organ cultures of the guinea-pig small intestine. Preparations of submucosa, with or without the myenteric plexus attached, were maintained in vitro for 3 to 5 days. Immunohistochemical labelling for peptides revealed that the cultured submucous plexus remained substantially intact and the immunoreactivity of cell bodies was well preserved. Substantial sprouting of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) was evident in submucous ganglia after 5 days in organ culture. Nerve fibers immunoreactive for substance P. somatostatin, 5-hydroxytryptamine or tyrosine hydroxylase were substantially depleted in submucous ganglia or perivascular nerves at 3 days and had virtually disappeared after 5 days in cultures of isolated submucosa. During intracellular recording from submucous neurons, action potentials were initiated by depolarizing current pulses in all neurons cultured with or without the myenteric plexus and muscle layers. Electrical stimulation of internodal strands evoked fast excitatory synaptic potentials (fast EPSPs) in nearly all neurons whether or not the myenteric plexus was present during the culture period up to 5 days. The removal of myenteric plexus and extrinsic nerves did not abolish fast EPSPs from submucous neurons, suggesting that some fast EPSPs may originate from neurons in the submucous plexus, although the possibility that new synapses formed by sprouting, or surviving axons severed from myenteric or sympathetic ganglia may have been functional, cannot be entirely excluded. This work demonstrates that the immunohistochemical and electrophysiological characteristics of submucous neurons are largely maintained in organ cultures of the submucosa.

Conducted depolarization in arteriole networks of the guinea-pig small intestine: effect of branching of signal dissipation.

1. Blood flow control requires co-ordinated activity among many branches of arteriole networks, which may be achieved by conduction of membrane potential changes between arteriolar smooth muscle cells and endothelial cells. 2. We investigated the effect of branching upon the passive conduction of electrical signals through the syncytium of electrically coupled cells in arteriole networks (n = 12) prepared from the guinea-pig submucosa. To describe the effect of branching on cable properties, the expansion parameter B was calculated (B = 1 for an unbranched cable; B > 1 with branching) for a point in each arteriole network based on anatomy. 3. An estimate of B(B') was also obtained by measuring the spread of depolarization caused by a high-K+ stimulus applied to one region. Membrane potential (-74 +/- 4 mV (+/- S.D.) at rest) was recorded from smooth muscle cells (verified with intracellular dye labelling). A micropipette containing 120 mM KCl was positioned at 150 micron increments along an arteriole (width, 50-75 microns) up to approximately 1.2 mm from a stationary recording site, producing stable depolarization which decreased as separation distance increased. The dissipation of depolarization with separation was greater when recording near branch origins rather than continuous segments. 4. B ranged in value from 0.99 to 2.28. In any one experiment, values of B and B' were correlated (correlation coefficient, r = 0.71; P < 0.05), but B' was consistently greater than B, and we discuss methodological factors which could lead to erroneously high values for B'. 5. For pooled electrophysiological data, depolarization decayed to 37% (1/e) of initial values in approximately 700 microns, consistent with B > 1. In contrast, the conduction of vasoconstriction and vasodilatation exceeds 2 mm in arteriole networks in previous studies. To explain this discrepancy, we suggest that active electrical events in cells of the arteriole wall augment passive electrical conduction during blood flow control.

Relative contributions of extracellular Ca2+ and Ca2+ stores to smooth muscle contraction in arteries and arterioles of rat, guinea-pig, dog and rabbit.

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca2+ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 mumol/L) with a transient contraction in Ca(2+)-free Krebs', reflecting Ca2+ release from PE-sensitive Ca2+ stores. After pretreatment with cyclopiazonic acid (CPA; 30 mumol/L) or thapsigargin (TSG; 1 mumol/L), putative SR Ca2+ pump inhibitors, the PE-induced contraction in a Ca(2+)-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 mumol/L), an agonist that enhances Ca2+ release from the SR, also reduced the PE contraction in a Ca(2+)-free solution. 3. CPA or TSG alone in the presence of extracellular Ca2+, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca2+ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca2+ fluxes across the plasma membrane predominates.

Actions of vasodilator nerves on arteriolar smooth muscle and neurotransmitter release from sympathetic nerves in the guinea-pig small intestine.

1. Brief constrictions of arterioles of the isolated submucosa of the guinea-pig small intestine were evoked by stimulation of the perivascular sympathetic nerves. Prior stimulation of vasodilator neurones in the submucosal nerve plexus greatly reduced the constrictor response to sympathetic stimulation. 2. Vasodilator nerve stimulation reduced both the amplitude and rate of decay of the excitatory junction potential (EJP) evoked in the arteriolar smooth muscle by sympathetic nerve stimulation. 3. Computer simulation of the effect of membrane resistance changes on the EJP amplitude indicated that the change in amplitude could not be explained by the fall in membrane resistance alone, suggesting that vasodilator nerve activity reduced neurotransmitter release from the sympathetic nerves.

Reduction of vasoconstriction mediated by neuropeptide Y Y2 receptors in arterioles of the guinea-pig small intestine.

Brief applications of a high-K+ solution were used to evoke transient constrictions of arterioles from the guinea-pig small intestine. Analogues of neuropeptide Y (NPY) selective for Y2-receptors reduced the constrictions, whereas NPY or a Y1-selective analogue potentiated the constrictions. We conclude that arteriolar smooth muscle has both Y1 and Y2 receptors, and suggest that Y2 receptors inhibit vasoconstriction by modulating the opening of voltage-sensitive Ca2+ channels. This may be related to the role of NPY that is present in some vasodilator nerves.

Vasodilatation and smooth muscle membrane potential changes in arterioles from the guinea-pig small intestine.

1. Dilatation of arterioles isolated from the guinea-pig small intestine was evoked by stimulation of a submucous ganglion and the application of acetylcholine, vasoactive intestinal peptide, galanin or dynorphin A. Changes in arteriole diameter and smooth muscle membrane potential were recorded simultaneously. 2. Ganglion stimulation caused vasodilatation and smooth muscle hyperpolarization that varied in both amplitude and time course from one arteriole to another. Vasodilatation could occur without hyperpolarization. 3. Vasodilatation caused by acetylcholine was accompanied by a rapidly developing hyperpolarization that began to decline before the maximum vasodilator effect had developed. 4. Vasoactive intestinal peptide caused dilatation without any change in smooth muscle membrane potential. 5. Galanin and dynorphin caused dilatation and a hyperpolarization of similar time course to the dilatation. 6. In 48% of arterioles tested the dilatation appeared to be mediated solely by acetylcholine. In 31% there was a cholinergic component, but no evidence for the involvement of acetylcholine in the remaining 21%. When the non-cholinergic dilatation occurred without a hyperpolarization we conclude that it was due to vasoactive intestinal peptide; otherwise it may have been due to either galanin or dynorphin.

Effects of neuropeptide Y and agonists selective for neuropeptide Y receptor sub-types on arterioles of the guinea-pig small intestine and the rat brain.

1. The actions of neuropeptide Y (NPY) and agonists selective for NPY receptor subtypes were examined on arterioles from the guinea-pig small intestine and the rat pia in order to characterize the receptors mediating the vasoconstrictor and potentiating effects of NPY. 2. A method was developed for measuring the potentiating effects of NPY in situations where it was not possible to obtain a full concentration-response relationship for the vasoconstrictor. NPY, 50 nM, had a greater potentiating effect on the guinea-pig intestinal arterioles than those from the rat pia. 3. NPY and the Y1-selective agonist, NPY[Leu31,Pro34], potentiated the constrictor responses to U46619 in both arterioles and responses to noradrenaline in the guinea-pig arterioles. There was marked desensitization of the potentiating effect, and cross-desensitization between NPY and NPY[Leu31,Pro34]. Both NPY and NPY[Leu31,Pro34] caused constriction of the rat pial arterioles but not of those from the guinea-pig intestine. 4. The Y2-selective agonist PYY(13-36) caused no potentiation or vasoconstriction and did not affect the potentiation by NPY or NPY[Leu31,Pro34]. 5. The potentiating and vasoconstrictor effects of NPY on these arterioles were mediated by Y1 receptors.

Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium.

Stimulation of the endothelial lining of arteries with acetylcholine results in the release of a diffusible substance that relaxes and hyperpolarizes the underlying smooth muscle. Nitric oxide (NO) has been a candidate for this substance, termed endothelium-derived relaxing factor. But there are several observations that argue against the involvement of NO in acetylcholine-induced hyperpolarization. First, exogenous NO has no effect on the membrane potential of canine mesenteric arteries. Second, although haemoglobin (believed to bind and inactivate NO (refs 11-15)) and methylene blue (which prevents the stimulation of guanylate cyclase) inhibit relaxation, neither has an effect on hyperpolarization. Finally, nitroprusside, thought to generate NO in vascular smooth muscle, relaxes rat aorta without increasing rubidium efflux. Nevertheless, nitrovasodilators, nitroprusside and nitroglycerin cause hyperpolarization in some arteries. NO might therefore be responsible for at least part of the hyperpolarization induced by acetylcholine. We now report that hyperpolarization and relaxation evoked by acetylcholine are reduced by NG-monomethyl-L-arginine, an inhibitor of NO biosynthesis from L-arginine. Thus NO derived from the endothelium can cause hyperpolarization of vascular smooth muscle, which might also contribute to relaxation by closing voltage-dependent calcium channels. Our findings raise the possibility that hyperpolarization might be a component of NO signal transduction in neurons or inflammatory cells.

Actions of neuropeptide Y on arterioles of the guinea-pig small intestine are not mediated by smooth muscle depolarization.

Neuropeptide Y was applied to arterioles of the submucosa of the guinea-pig small intestine while arteriole diameter and smooth muscle membrane potential were monitored. Neuropeptide Y (50 nM-1 microM) caused no smooth muscle depolarization, and caused a small constriction in only 15 out of 38 arterioles studied. 50 nM Neuropeptide Y increased the amplitude of constriction caused by noradrenaline or brief trains of nerve stimulation, showing that it potentiated the effects of vasoconstrictors as it does in other arteries. The factor by which the amplitude was increased was greatest for small constrictions. Neuropeptide Y reduced the amplitude of the excitatory junction potential, suggesting that it decreased neurotransmitter release. These results show that the potentiating action of Neuropeptide Y does not depend on smooth muscle depolarization.

Vasodilatation of arterioles by acetylcholine released from single neurones in the guinea-pig submucosal plexus.

The nervous control of arterioles in the guinea-pig submucosal plexus was studied. Outside diameters of arterioles were recorded using a video-monitoring system. Changes in arteriolar diameter in response to electrical stimulation of single neurones or ganglia in the plexus were measured. 2. When the arteriole was pre-constricted with the prostaglandin analogue U46619 or with phenylephrine, electrical stimulation (2-20 Hz, 10 s) of a ganglion dilated the blood vessel. This vasodilatation was abolished by tetrodotoxin or by cutting the fine nerve strands running between the ganglion and the arteriole. 3. The vasodilatations caused by ganglionic stimulation were blocked by the muscarinic antagonists atropine, pirenzepine, (11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-)-one (AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) and hexahydrosilodifenidol (HSDF). IC50 values for the inhibition of nerve-evoked vasodilatation by pirenzepine, AFDX-116 and HSDF were 500 nM, 4 microM and 25 nM respectively. Physostigmine (1 microM) increased the dilatation by 90%. 4. Muscarine dilated all submucosal arterioles; the concentration causing half-maximum effects was 200 nM. Muscarinic vasodilatations were inhibited by pirenzepine, AFDX-116, and HSDF in a competitive manner; dissociation equilibrium constants determined by Schild analyses were 125 nM, 1.3 microM and 4 nM respectively. 5. Gossypol, an irreversible inhibitor of the production of endothelium-derived relaxing factor (EDRF), did not reduce the vasodilatation produced by either ganglionic stimulation or muscarine in submucosal arterioles. 6. Intracellular recordings were made from submucosal neurones and action potentials were elicited by depolarizing current pulses (10 ms duration, 10 Hz/10 s). In seven neurones vasodilatation was associated with intracellularly evoked action potentials; this vasodilatation was blocked by pirenzepine. Cell bodies of reidentified vasodilator neurones were subsequently shown to contain immunoreactive choline acetyltransferase. 7. These results show that cholinergic neurones in the submucosal plexus project to submucosal arterioles and that they release acetylcholine onto muscarinic receptors to produce vasodilatation. The muscarinic receptor activated by nerve-released acetylcholine is the M3 subtype and its location appears to be on the vascular smooth muscle rather than the endothelium.

Response of the rat tail artery to prolonged exposure to noradrenaline.

1. The contractions of the rat tail artery in response to noradrenaline applied for 30 min periods were recorded under conditions that potentiate the vascular escape phenomenon (spontaneous partial relaxation in the continued presence of a vasoconstrictor) in smaller arteries. 2. The conditions were elevated temperature (from 32-37 degrees C), 500 nM forskolin and 10 microM 3-isobutyl-1-methyl xanthine. 3. None of these conditions caused any change in the time-course of constriction in response to noradrenaline, or produced any evidence of vascular escape in this large artery.