Electrical Coupling between the Myenteric Interstitial Cells of Cajal and Adjacent Muscle Layers in the Guinea-Pig Gastric Antrum

Intracellular recordings were made from short segments of the muscular wall of the guinea-pig gastric antrum. Preparations were impaled using two independent microelectrodes, one positioned in the circular layer and the other either in the longitudinal layer, in the network of myenteric interstitial cells of Cajal (ICC _my ) or in the circular layer. Cells in each layer displayed characteristic patterns of rhythmical activity, with the largest signals being generated by ICC _my . Current pulses injected into the circular muscle layer produced electrotonic potentials in each cell layer, indicating that the layers are electrically interconnected. The amplitudes of these electrotonic potentials were largest in the circular layer and smallest in the longitudinal layer. An analysis of electrical coupling between the three layers suggests that although the cells in each layer are well coupled to neighbouring cells, the coupling between either muscle layer and the network of ICC _my is relatively poor. The electrical connections between ICC _my and the circular layer did not rectify. In parallel immunohistochemical studies, the distribution of the connexins Cx40, Cx43 and Cx45 within the antral wall was determined. Only Cx43 was detected; it was widely distributed on ICC _my and throughout the circular smooth muscle layer, being concentrated around ICC_IM, but was less abundant in the circular muscle layer immediately adjacent to ICC _my . Although the electrophysiological studies indicate that smooth muscle cells in the longitudinal muscle layer are electrically coupled to each other, none of the connexins examined were detected in this layer.     

Rhythmicity in arterial smooth muscle

Many arteries and arterioles exhibit rhythmical contractions which are synchronous over considerable distances. This vasomotion is likely to assist in tissue perfusion especially during periods of altered metabolism or perfusion pressure. While the mechanism underlying vascular rhythmicity has been investigated for many years, it has only been recently, with the advent of imaging techniques for visualizing intracellular calcium release, that significant advances have been made. These methods, when combined with mechanical and electrophysiological recordings, have demonstrated that the rhythm depends critically on calcium released from intracellular stores within the smooth muscle cells and on cell coupling via gap junctions to synchronize oscillations in calcium release amongst adjacent cells. While these factors are common to all vessels studied to date, the contribution of voltage-dependent channels and the endothelium varies amongst different vessels. The basic mechanism for rhythmical activity in arteries thus differs from its counterpart in non-vascular smooth muscle, where specific networks of pacemaker cells generate electrical potentials which drive activity within the otherwise quiescent muscle cells.     

Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses

It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca²⁺-activated K⁺ channels channels by InsP₃-induced Ca²⁺ release from the endoplasmic reticulum and/or store-operated Ca²⁺ entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.     

Non-invasive assessment of corneal endothelial permeability by means of electrical impedance measurements

The permeability of the corneal endothelial layer has an important role in the correct function of the cornea. Since ionic permeability has a fundamental impact on the passive electrical properties of living tissues, here it is hypothesized that impedance methods can be employed for assessing the permeability of the endothelial layer in a minimally invasive fashion. Precisely, the main objective of the present study is to develop and to analyze a minimally invasive method for assessing the electrical properties of the corneal endothelium, as a possible diagnostic tool for the evaluation of patients with endothelial dysfunction. A bidimensional model consisting of the main corneal layers and a four-electrode impedance measurement setup placed on the epithelium has been implemented and analyzed by means of the finite elements method (FEM). In order to obtain a robust indicator of the permeability of the endothelium layer, the effect of the endothelium electrical properties on the measured impedance has been studied together with reasonable variations of the other model layers. Simulation results show that the impedance measurements by means of external electrodes are indeed sufficiently sensitive to the changes in the electrical properties of the endothelial layer. It is concluded that the method presented here can be employed as non-invasive method for assessing endothelial layer function.     

Role of Cellular Orientation in Electrical Coupling Between Gastrointestinal Smooth Muscle

Electrical fields produced during depolarization as well as low resistance pathways through gap junctions have been proposed as electrical coupling mechanisms serving to coordinate electrical control activity in gastrointestinal smooth muscle. The differing orientations of the longitudinal and circular muscle layers offer many possible configurations for cells coupled by electrical fields. The boundary element method is used to investigate coupling, with respect to both gap junctions and field effects for ellipsoidal and cylindrical cells. Physiological considerations allow the possibility of aggregates of cells with coordinated electrical activity. The effect of multiple source cells on field coupling is also modeled. Results indicate that even small gap junctional conductances are effective for coupling of smooth muscle and that field coupling is most efficacious when the ellipsoidal cells are coupled side by side and when cylindrical cells are coupled end to end. © 1998 Biomedical Engineering Society. PAC98: 8722Jb, 8710+e, 0260Lj, 8750-a     

Ethanol uncouples dentate granule neurons by increasing junctional resistance: a multineuronal system model approach.

The effects of an acute intoxicating concentration of ethanol (50 mM) on the electrotonic membrane properties of hippocampal dentate granule neurons were studied using a system model incorporating electrotonic coupling between neurons. Uncoupling of cells by other alcohols has been shown in several tissues. The system model allows a quantitative estimation of the changes in coupling and other neuronal electrotonic properties. The input impedance of a neuron was measured from the voltage decay of a short hyperpolarizing current pulse. An analytic expression of the input impedance has been written incorporating somatic, dendritic, and electrical coupling parameters. Using this particular current stimulation, the modelling results showed that ethanol selectively increased the junctional resistance by more than 2.5 times, hence uncoupling the neurons. A 30% increase in the final time-constant, tau 0, was also obtained from the voltage transient. Other parameters were not significantly affected. A neuronal model without electrotonic coupling to other neurons gave rise to physiologically impossible values for the membrane resistance and capacitance. With resistive and capacitive coupling in the model, uncoupling did not occur with ethanol. It is concluded that ethanol uncouples neurons by increasing the effective gap junctional resistance in dentate granule neurons.     

Localization of myosin II regulatory light chain in the cerebral vasculature

The cytoskeleton of cerebral microvascular endothelial cells is a critical determinant of blood-brain barrier (BBB) function. Barrier integrity appears to be particularly sensitive to the phosphorylation state of specific residues within myosin regulatory light chain (RLC), one of two accessory light chains of the myosin II motor complex. Phosphorylation of myosin RLC by myosin light chain kinase (MLCK) has been implicated in BBB dysfunction associated with alcohol abuse and hypoxia, whereas dephosphorylation may enhance BBB integrity following exposure to lipid-lowering statin drugs. Using immunohistochemistry we provide evidence of widespread myosin II RLC distribution throughout the cerebral vasculature of the mouse. Light microscopy revealed immunolocalization of myosin II RLC protein in the endothelium of brain capillaries, the endothelial cell layer of arterioles and in association with venules. Immunolabeling of myosin RLC in non-muscle endothelial cells could be distinguished from myosin RLC immunoreactivity associated with the smooth muscle layer of the tunica media in larger muscular arterioles. These findings support an emerging role for myosin II RLC as a component of the actomyosin cytoskeleton of cerebral endothelial cells with the potential to contribute to the selective vulnerability of the brain in vivo.     

Electrical resistance of arterioles and venules in the hamster cheek pouch

The electrical resistance of the vascular endothelium was determined on single microvessels in the hamster cheek pouch in order to obtain information about this variable in a mammalian preparation. So far, the technique has only been applied to frog microvessels. The technique consists of injection of current into the vascular lumen via a microelectrode and recording of the ensuing intravascular potential distribution by a second microelectrode. Cable theory was used for the analysis. The average diameter of the vessels under study was 41 micron for arterioles and 28 micron for venules. The average resistance of the vessel wall at 37 degrees C was 19 omega cm2 and 3.3 omega cm2, respectively. For the venules this is somewhat lower than what has been recorded on muscle capillaries (Olesen & Crone 1983) in the frog at room temperature, whilst the values on arterioles are rather similar. The calculated sodium permeabilities, PNa+, were for arterioles 4 X 10(-5) cm X s-1 and for venules 23 X 10(-5) cm X s-1. The high permeability values for arterioles and venules indicate that the vascular exchange function may not be limited to capillaries only.     

Aligned 3D human aortic smooth muscle tissue via layer by layer technique inside microchannels with novel combination of collagen and oxidized alginate hydrogel

Tissue engineering of the small diameter blood vessel medial layer has been challenging. Recreation of the circumferentially aligned multilayer smooth muscle tissue has been one of the major technical difficulties. Some research has utilized cyclic stress to align smooth muscle cells (SMCs) but due to the long time conditioning needed, it was not possible to use primary human cells because of expeditious senescence occurred . We demonstrate rapid buildup of a homogeneous relatively thick (30-40 μm) aligned smooth muscle tissue via layer by layer (LBL) technique within microchannels and a soft cell-adhesive hydrogel. Using a microchannelled scaffold with gapped microwalls, two layers of primary human SMCs separated by an interlayer hydrogel were cultured to confluence within the microchannels. The SMCs aligned along the microchannels because of the physically constraining microwalls. A novel double layered gel consisting of a mixture of pristine and oxidized alginate hydrogel coated with collagen was designed to place between each layer of cells, leading to a thicker tissue in a shorter time. The SMCs penetrated the soft thin interlayer hydrogel within 6 days of seeding of the 2nd cell layer so that the entire construct became more or less homogeneously populated by the SMCs. The unique LBL technique applied within the micropatterned scaffold using a soft cell-adhesive gel interlayer allows rapid growth and confluence of SMCs on 2D surface but at the same time aligns the cells and builds up multiple layers into a 3D tissue. This pseudo-3D buildup method avoids the typical steric resistance of hydrogel embedding.     

The Fate of Mural Thrombi Produced by Injury in the Ear Artery of the Rabbit

By exposing the central artery of the ear of a rabbit through a skin incision it is possible regularly to produce platelet thrombi in the lumen by a unipolar electrical stimulus. By closing the skin incision the fate of such thrombi has been followed up to 50 days. One to 2 hours after injury endothelium has disappeared completely and the media is destroyed. A thrombus consisting of amorphous material and platelets is present on the luminal surface. During repair endothelium grows over the thrombus and smooth muscle cells migrate into it. These cells gradually become mature, a stage reached at about 15 days, at which time elastic fibres and collagen appear between the muscle cells. At 50 days there are several layers of smooth muscle cells interspersed with collagen and elastic fibres interleaved between endothelium and internal elastic lamina.     

Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling.

1. Segments of mouse or rat pancreas were placed in a flow cell through which physiological salt solutions of varying composition were pumped at a constant rate. Intracellular recordings of membrane potential, resistance and electrical time constant were made from the acini using fine glass micro-electrodes. In some experiments two micro-electrodes were inserted into two acinar cells within the same acinus to assess directly cell to cell coupling. The concentration of amylase in the effluent was measured continuously. 2. Electrical coupling between two acinar cells was observed when the tips of the two micro-electrodes were less than 50 mum from each other. The coupling ratio was close to 1. Acetylcholine (ACh) always evoked depolarization of exactly the same amplitude in two coupled cells and reduced the amplitude of current-pulse induced membrane potential changes in both cell simultaneously. 3. Stimulation with ACh caused an immediate increase in amylase output. Replacement of superfusion fluid Na by Tris or Cl by sulphate abolished ACh-evoked increase in amylase release, but the subsequent reintroduction of Na or Cl caused an increase in amylase release of a magnitude similar to what was normally observed following stimulation. 4. Omitting Ca from the superfusion fluid and adding EGTA rapidly depolarized the acinar cell membrane, reduced the input resistance and caused a marked reduction in amylase secretion. During exposure to a Ca-free, EGTA containing solution a marked increase in amylase release occurred following maximal ACh stimulation. 5. Addition of small amounts of Mg, Ca or Mn to a Ca-, Mg-free solution caused an increase in membrane potential, input resistance and electrical time constant and markedly increased amylase release. The effect on the electrical parameters was reversed in the absence of extracellular Na while extracellular Na was of no importance for the effect on amylase release. 6. The effect of ACh on amylase was enhanced during superfusion with a fluid containing 20 mM-Ca. The presence of Mn (5 mM) in an otherwise normal control had no effect on ACh-evoked release. 7. These results show that ACh acts on the acinus by reducing the surface cell membrane resistance. It is suggested that the ACh-receptor interaction causes a release of Ca from the surface cell membrane and that the concentration of Ca in the surface cell membrane determines the specific membrane resistance particularly for Na. The release of Ca to the cytosol activates exocytosis while the Na influx is of importance for acinar fluid secretion. The effect of ACh on amylase secretion can be mimicked by agents displacing membrane-bound Ca (Mg, Ca, Mn).     

Dependency of endothelial cell function on vascular smooth muscle cells in guinea-pig mesenteric arteries and arterioles.

Using guinea-pig mesenteric arteries and arterioles, we investigated the membrane potential of endothelial cells at rest and during application of acetylcholine (ACh) with and without the smooth muscle layers attached. When smooth muscle and endothelial layers were in close apposition, the resting membrane potentials of the two types of cells were closely related and were slightly more negative in the smooth muscle cells than in the endothelial cells. Once the endothelial layer was separated from the smooth muscle layer, the endothelial cells depolarized (the average, -4.2 mV). In the isolated endothelial layer, ACh did not induce a membrane hyperpolarization as expected, but did induce a quick depolarization soon after conventional whole-cell recording was started. However, as the pipette solution (high K+) gradually diffused into the endothelial layer, the membrane response to ACh gradually changed toward hyperpolarization. ACh-induced hyperpolarization was also observed after incubating preparations in a high-potassium bath solution. Our results indicate that vascular smooth muscle cells and endothelial cells are influencing each other as a functional unit and that the endothelial cells rely on the smooth muscle cells for their intracellular ionic composition and resting membrane potential.     

Angiogenesis in the postovulatory primate endometrium: the coiled arteriolar system

The postovulatory period in the primate endometrium of the menstrual cycle is characterized by rapid growth of the coiled arterioles. A great variety of developing microvascular components occurs among a well-differentiated microvasculature of coiled arterioles, capillaries, and venules. Endometrial biopsies were obtained by hysterotomy during progesterone dominance at 5, 6, 7, 10, 12, and 14 days following the peak of the estrogen surge as determined by serum radioimmunoassay. Arteriolar ultrastructural differentiation is remarkably similar on each of these days. Ultrastructural evidence of elastogenesis in the extracellular matrix adjacent to certain endothelial tubes provides the initial sign of coiled arteriolar formation. The cellular primordia of the tunica intima and media are identified by spatial location and glycogen storage in smooth muscle cells. Endothelial projections span the incipient internal elastic membrane to make contact with the surfaces of the innermost vascular smooth muscle cells. Subsequent arteriolar differentiation centers on formation of a muscular media composed of 1 or 2 muscle layers separated by a spiraling lamellar elastic matrix that appears initially between the endothelial tube and the first muscle layer. Vascular smooth muscle cells are highly branched and linked across the elastic matrix by surface contacts. Definitive coiled arterioles consist of interlinked endothelial and smooth muscle cells within a thick, spiraling elastic matrix that provides flexibility for rapid changes in shape. Progressive differentiation of coiled arterioles continues up to the premenstrual stage. This abundant angiogenesis may reflect preparation and maintenance of a suitable uterine environment for the possibility of implantation and pregnancy during each menstrual cycle.     

Scanning electron microscopy of mouse muscle microvasculature

The surface morphology of the microvasculature from mouse skeletal muscle was studied by scanning electron microscopy. Cell surfaces were exposed by digesting away extracellular collagen and other matrix by a simple HCl treatment. Four distinct subdivisions of the microvasculature (arterioles, precapillary arterioles, capillaries, and venules) were identified based on marked differences in surface features. Arterioles of 20–10 μm diameter had a discontinuous, single layer of smooth muscle cells encircling the vessel. These smooth muscle cells had an uneven surface with shallow grooves and depressions that were often oriented parallel to the longitudinal cell body axis. The underlying arteriolar endothelial surface was also rough with long ridges separating shallow furrows that were oriented parallel to the vessel length. As the arteriolar size decreased, the perivascular cell were found further apart, they became smooth surfaced, and were oriented preferentially parallel to the vessel. The endothelium of the precapillary arterioles, as well as, capillaries and venules had smooth surfaces. Venules had a discontinuous layer of flat, smooth surfaced pericytes. Morphologically distinct groups of smooth muscle cells (i.e., precapillary sphincters) were not found. Although pericytes normally associated with capillaries and other vessels were often removed during tissue processing, most cells and their surface feature were generally well preserved.     

Large arterioles in the control of blood flow: role of endothelium‐dependent dilation

Although it is generally assumed that small arterioles form the major site of vascular resistance, microcirculatory studies revealed that 40–55% of the total network resistance can reside in large arterioles and small arteries. Thus, the mechanisms that control smooth muscle tone in these vessels have a major impact on the overall conductance of the vascular network. These control mechanisms are different from those in small arterioles: Aside from an apparently reduced sensitivity to metabolites, the large resistance vessels are normally too far away from the capillary areas which they feed to be reached by diffusing metabolites from dependent cells within a reasonable period of time. Rather, recent intravital microscopic studies suggest that large resistance vessels are under tight control of endothelial factors such as nitric oxide and endothelium‐derived hyperpolarising factor (EDHF). Nitric oxide opposes myogenic constrictions of large arterioles that potentially would impair tissue perfusion and oxygenation. Moreover, nitric oxide and EDHF play an important role in the co‐ordination of large and small resistance vessel behaviour that is pivotal for the adaptation of blood flow to altered tissue oxygen demands.     

Current-voltage relationships in the crystalline lens.

1. Electrical coupling between the cells of the crystalline lens of the frog eye was studied using two intralenticular micro-electrodes, one to pass current and one to record potential. In most experiments, both electrodes were placed just inside the posterior surface of the lens at a depth of approximately 200 mum from the surface. Step functions of current were applied and the time course of the resulting change in voltage was measured at many different electrode separations. 2. The voltage change has both a fast component, which occurs only locally in the region close to the current passing micro-electrode, and a slow component, which is spatially uniform, independent of distance from the current micro-electrode. 3. This behaviour is predicted by an electrical model of a single large spherical cell, and so that model can be used to analyse our data. 4. The resistivity of the lens 'interior' (both cytoplasm and coupling resistivity) is 625 omega cm; the resistance of the lens 'membrane' is 2751 omega cm2. 5. The data and analysis help to reconcile discrepancies between previous measurements of the electrical properties of the lens and show clearly that there is substantial electrical coupling from cell to cell. The method should allow investigation of the role of electrical coupling in cataract formation in the crystalline lens.     

Non-homogeneous blood flow distribution in the rabbit tenuissimus muscle Differential control of total blood flow and capillary perfusion

Structural and functional relationships underlying the blood flow distribution in the rabbit tenuissimus muscle were examined by means of intravital microscopy. A majority of the main feeding arterioles (transverse arterioles) continued into adjacent connective tissue, after giving off branches (terminal arterioles) within the muscle tissue to supply the muscle capillaries. The transverse arterioles thus supplied two vascular areas, although the major part of the arteriolar flow, under normal resting conditions, was distributed to the muscle capillaries—a flow fraction over which the terminal arterioles exerted ultimate control. The fractional distribution of the blood flow between muscle and connective tissue was determined by the relative contributions of the transverse and terminal arterioles to the vascular resistance. These arteriolar segments showed a differential response to an increase in oxygen availability (elevated ambient pO₂), resulting in a total reduction of muscle capillary flow, but no concomitant change in the flow to connective tissue. A decrease in perfusion pressure, on the other hand, led to similar flow changes in the muscle and connective tissue circulation, which was attributed to proportionate resistance changes in the transverse and terminal arterioles. Differences between the larger transverse and smaller terminal arterioles in their sensitivity to various stimuli may form a functional basis for a differential control of arteriolar blood supply and capillary perfusion in this muscle.     

Current spread in the smooth muscle of the rabbit aorta

1. The electrical responses of the smooth muscle cells of the rabbit aorta to both extracellular and intracellular stimulation were studied using the partitioned chamber and Wheatstone bridge method.2. No spontaneous electrical activity was recorded when the tissue was soaked in either isotonic or hypertonic Krebs solutions, and strong depolarizing currents also failed to trigger action potentials in either solution.3. The circular muscle of the aorta has cable properties. Mean values in isotonic Krebs solution were 2.1 mm for space constant and 433 msec for time constant.4. The input resistance (mean 12 MOmega) measured with the Wheatstone bridge method was considerably smaller than that calculated from values measured with the partitioned chamber method.5. Electrotonic potentials could be recorded from the smooth muscle of ;injury bundles' although their amplitude was smaller than that from the intact bundle.6. High concentrations of noradrenaline readily induce oscillatory potentials from the aorta in both isotonic and hypertonic Krebs solutions. It was estimated by simultaneous recording with two micro-electrodes that noradrenaline-induced oscillatory potential can conduct in both longitudinal and transverse directions of the smooth muscle.7. These results suggest that the smooth muscle of the aorta behaves like a syncytium or single unit muscle and activation of cells on the inner surface of the media can be induced both by electrotonic current spread and by propagation of oscillatory potentials from the outer cells directly activated by the transmitter.     

The fine structure of the interstitial tissue of the rat prostate

The structure of the interstitial tissue of the rat prostate has been studied using the light and electron microscopes in an attempt to determine the role of the fibromuscular stroma in the normal functioning of the gland. Smooth muscle cells and fibroblasts are the most numerous cell types. They are accompanied by macrophages, mast cells, and undifferentiated cells of low electron density. Smooth muscle cells have cytoplasmic protrusions that extend into corresponding depressions in adjacent muscle cells, and, at these points, the intercellular space is narrowed to 150–2000 Å. Smooth muscle cells and fibroblasts are arranged in parallel in septa between adjacent epithelial alveoli and form a sheath around each alveolus. Proceeding peripherally from the epithelium into the interstitial tissue, this sheath is composed of a layer of one or two highly flattened fibroblasts and a parallel layer of smooth muscle cells, followed subsequently by additional layers of fibroblasts and smooth muscle that merge into the remainder of the interstitial tissue. Most of the capillaries have an uninterrupted endothelium, but in some regions endothelial fenestrations are present. Unmyelinated axons contain aggregations of small granular and agranular vesicles. Vesicles are found in axons at distances up to several thousand angstroms from muscle cells and in axons that approach to within 150–200 Å of smooth muscle cells. In some cases an axon lies in a deep depression in the surface of a muscle cell. The type of innervation and variety of intercellular contact between muscle cells is discussed in relation to probable physiological characteristics of the tissue.