Temperature-dependence of45Ca fluxes and contraction in vascular smooth muscle cells of rabbit ear artery

The effect of cooling from 35 to 20° C on the⁴⁵Ca-exchange and on the contractile response of rabbit ear artery has been investigated.The amplitude of the contraction induced by K-depolarization at 20° C is reduced to about 60% of its value at 35° C, whereas the response to noradrenaline is not significantly affected.Cooling induces a 2 to 4-fold reduction of the⁴⁵Ca-efflux rate. This effect also occurs in Ca-free medium and in solutions containing 1 mM La. It also occurs in Na-free medium and in tissues in which the transmembrane Na-gradient has been reduced.At 20° C, the⁴⁵Ca-influx in unstimulated tissues and in K-depolarized preparations is significantly lower than at 35° C. in Ca-depleted tissues, i.e. tissues in which the noradrenaline-sensitive Ca-store has been emptied by a stimulation with the agonist in Ca-free solution, the⁴⁵Ca-influx is not significantly affected by cooling.The gradual depletion of the noradrenaline-sensitive Ca-store in Ca-free solutions is at 20° C much slower than at 35° C. The amount of Ca released by noradrenaline is not affected by cooling, whereas for the same amount of Ca released the contractile response is higher at 20° C.These findings indicate that temperature affects the transmembrane Ca-extrusion and the Ca-influx through voltage-dependent channels. The properties of the noradrenaline-sensitive Ca-store are less sensitive to temperature.     

Ionic fluxes in rat uterine smooth muscle.

1. The K+, Na+ and Cl-fluxes from the oestrogen-stimulated rat uterine smooth muscle were measured using radioactive tracers. 2. The cellular compartment contained a concentration of K+ of 173-6 mM which exchanged at a rate of 5-9 x 10(-12) mol.cm-2.sec-1. 3. Cl- exchanged at a rate of 6-9 x 10(-12) mol.cm-2.sec-1 from a cellular compartment having a concentration of 39-3 mM. 4. The methods used for the evaluation of Na+ movements over-estimate both influx and efflux values. If an average value of flux of 9-2 x 10(-12) mol.cm-2.sec-1 is considered we obtain PNa+/PK+ ratios of 2-4 (-42 mV) or 1-3 (-57-6 mV), which are too high and do not correspond to electrophysiological evidence. 5. The relative permeabilities PCl-/PK+ in the case of a membrane potential of -42-0 mV could be 0-8, or 1-3 in the case of a membrane potential of -57-6 mV. 6. Both conductances GK+ and GCl- seem to play an important role in determining membrane conductance.     

Longitudinally orientated smooth muscle cells in rabbit arteries

Intima formation in vessels, spontaneous or experimentally induced, is generally characterized by the presence of longitudinally orientated smooth muscle cells (LSMC). During an experiment of neo-intima induction in carotid arteries in rabbits, by application of a nonconstrictive silastic cuff, a study was performed to investigate the presence of LSMC in the systemic and pulmonary circulations, in both elastic and muscular arteries. Three patterns could be distinguished: intimai cushions in muscular arteries, single or small groups of LSMC in the intima in elastic and larger muscular arteries, and intra-medially located layers or columns of LSMC in the aorta, the pulmonary artery, at the bifurcation of the aorta and around orifices of branches. In order to understand this peculiar orientation a biomechanical approach was used: this showed that near the lumen the circumferential stress is 4.5 times higher than the longitudinal. Because the cell surface of the smooth muscle cells exposed to this stress per unit vessel length is much less in the longitudinal than in the circular direction we conclude that the LSMC align in the direction which allows them to cope most effectively with the mechanical stresses.     

Graded activation in rabbit mesotubarium smooth muscle.

The outer margin of the mesotubarium superius, an accessory ligament from the femal rabbit reproductive system, contains a long and slender bundle of smooth muscle fibers well aligned with the long axis of the tissue. Very little connective tissue is present. Massive alternating-current electrical field stimuli (variable in frequency, amplitude, and duration) applied to isolated mesotubaria from mature, nonpregnant animals produced contractions in which isometric force (P) and its first derivative (dP/dt) could be continuously graded; an optimum existed for stimulus frequency and amplitude, but not for its duration. Twitchlike contractions could not be produced. Isometric contractions in which P was plotted against dP/dt to generate a phase-plane trajectory were used to predict the ultimate (time = infinity) force (Po) in graded contractions; this value agreed with the Po derived from isotonic force-velocity curves fitted to the Hill equation. Quick stretches applied during the rise of contractile force revealed a slow onset of the ability to bear the Po force.     

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.     

Ouabain-sensitive ion fluxes in the smooth muscle of the guinea-pig''s taenia coli.

1. Tissues with raised intracellular Na levels, produced by incubation in K-free media, were used throughout. The uptake of 42K by these Na-loaded tissues was followed for 10 min in the presence and absence of 1-37 X 10(-4) M ouabain, this being sufficient to inhibit Na pumping maximally. Subtraction of the uptake seen in the presence from that seen in the absence of ouabain gave estimates of the pumped ouabain-sensitive K uptake. 2. In Na-free (MgCl2) medium this depended on the [K]0 in a sigmoidal fashion with a half maximal [K]0 for activation of some 4mM. The maximal uptake of K was 3 m-mole/kg.min corresponding to a transmembrane flux of some 12-5 p-mole. cm-2.sec-1. 3. In the presence of Na the K activation curve became more obviously sigmoid and higher concentrations of K were needed to achieve a given active K influx. The results were well fitted by assuming that Na and K competed for two identical, non-interacting sites on the external pump face. 4. Addition of K during the efflux of 24Na into a Na-free (MgCl2) medium led to an increased rate of tracer loss. The magnitude of this increase depended on the [K] used in a hyperbolic fashion and it was abolished by addition of ouabain. The [K] causing half-maximal activation of ouabain-sensitive Na efflux was in the order of 1-2 mM. 5. When the [K] in the uptake media was 1-5 mM; Na, Li, Rb and Cs all inhibited ouabain-sensitive K uptake, the order of effectiveness being Rb greater than Cs greater than Na greater than Li. With a E1TKA10 OF 0-15 MM low concentrations of Cs and Rb were shown to stimulate K uptake. Such an effect is predicted by assuming two ion binding sites on the pump's outer face, and that the pump can translocate mixtures of K and either Rb or Cs...     

Electrogenic sodium pumping in rabbit small intestinal smooth muscle.

Oscillations in the activity of an electrogenic Na pump has been suggested as the ionic mechanism underlying the intestinal control potential (slow wave). We investigated the electrogenicity of this pump in rabbit jejunal smooth muscle. Potassium admission to Na-rich tissues caused a large increase in membrane potential which after 10--20 min decreased toward values comparable with those of normal tissues. This hyperpolarization far exceeded EK and could be prevented by cooling or by ouabain. No hyperpolarization occurred upon K admission to Li-rich tissues in the absence of Na. Thus, the pump in this tissue can operate electrogenically. Goldman's equation was modified so as to account for the pump's contribution to the membrane potential. Using this equation, the calculated contribution of the pump, under normal "steady-state" conditions, is unlikely to exceed a few millivolts. It is concluded that although the pump in this tissue can be electrogenic, its contribution may be smaller than that required if the intestinal control potential resulted from rhythmic turning off and on of the electrogenic Na pump.     

Elastin distribution in the myometrial and vascular smooth muscle of the human uterus

Magnetic resonance imaging and transvaginal ultrasonography in women of reproductive age suggest that the myometrium consists of inner and outer layers. It was hypothesized that these structural and functional differences in the myometrium might be associated with a variation in elastin distribution. Fifty-one hysterectomy specimens representing all phases of the normal menstrual cycle were studied by immunocytochemistry, orcein staining and image analysis. Elastin was present within the outer myometrial smooth muscle, but was less widely distributed in the inner smooth muscle. Immunoreactivity and staining were observed in the myometrial arteries and arterioles and within the basal portions of endometrial arterioles. Elastin was also present in perivascular tissue, particularly near the large vessels. More extravascular (i.e. perivascular and smooth muscle) elastin was present in the outer myometrium in all cases, although no distinct layering was observed. Semi-quantitative analysis of the elastin distribution in 11 full thickness specimens demonstrated a decreasing gradient from outer to inner myometrium rather than distinct layering. Contrary to previous reports, these data suggest that the external region of the myometrium is more elastic than the inner region and that elastin is found throughout the arteriolar tree of the human uterus.     

Rectification in the smooth muscle cell membrane of rabbit aorta.

1. The current-voltage relation of the smooth muscle cell membrane of rabbit aorta was determined by the partition method. 2. No anomalous rectification was observed in any of the following solutions: normal Krebs, Na free choline, Na sulphate, and high K-Na free sulphate. 3. Delayed rectification was seen on application of depolarizing current in both normal Krebs solution and Na free choline solution. 4. High concentration of K made the steady-state current-voltage relation almost linear in a voltage range of about 0 to -20mV. This effect, and steady-state cathodal rectification which was seen in physiological solution, could be explained qualitatively by constant field theory without involving channels capable of anomalous rectification. 5. A slow decrease in K conductance, during application of large and long-lasting hyperpolarizing currents, which occurs in skeletal muscle and is attributed to the tubule system, was never observed in the arteries either in Krebs, Na-free choline, or Na sulphate solution.     

Age-related changes in adrenergic neuronal function of rabbit vascular smooth muscle

The adrenergic control of vascular smooth muscle was compared in young and adult rabbits using a variety of in vitro techniques. Norepinephrine (NE) content and accumulation of ³H-NE were not different in blood vessels from the two age groups. In contrast, stimulation-evoked release of endogenous NE was reduced by 40–60% in vessels from the aged animals. Functional studies of smooth muscle contractions were carried out using isolated ring segments of the ear artery. There were no differences in the resting force-response relationship between vessels from young and adult rabbits. Maximum contractile responses to nerve stimulation, NE or KCl were not different in vessels from the two age groups, nor was the NE ED₅₀. However, blockade of the neuronal uptake system with desmethylimipramine produced a greater shift in the NE concentration-response curve in vessels from the young animals compared to the shift in vessels from adult animals. This observation reflects a decline in neuronal NE uptake with age. Although maximal contractile responses to transmural nerve stimulation at 16 Hz were unchanged, responses to stimulation at lower frequencies were reduced in vessels from adult rabbits, an effect which was also enhanced when an antagonist of neuronal uptake was present. Thus, there is a decline in function of adrenergic nerves in adult animals, reflected in a decrease in stimulation-evoked NE release and a decrease in norepinephrine uptake revealed by functional studies. These two effects tend to balance each other, so that there is a small decrease in contractile response to adrenergic nerve stimulation which is exacerbated when neuronal uptake mechanisms are blocked.     

Three-dimensional structure of dense bodies in rabbit renal artery smooth muscle

In this report, we present a three-dimensional computer assisted reconstruction study from serial thin sections through a rabbit renal artery smooth muscle cell. In a series of 32 consecutive thin (100-nm) sections, one longitudinally oriented cell was followed and photographed in alternating sections. The profiles of the cell surface and dense bodies were reconstructed from these 16 planes and the distribution, size, shape, and spatial relationships among these components was examined. The reconstructed images showed that the cell decreases in diameter from its widest region in the center to the two ends in a step-wise taper. Within the cell, dense bodies are numerous. Relative to the cell axes, a membrane associated dense body (MDB) can be ≤3.5 μm long, 0.25 μm wide, and may extend up to 2 μm in depth. While the MDB profile in one section may be aligned with the long axis of the cell, in an adjacent section the same dense body may appear almost circular or wedge shaped. The same is true of cytoplasmic dense bodies (CDBs). Compared with MDBs, CDBs are smaller in all dimensions. Some, but not all, CDBs line up in strings oriented with the long axis of the cell. The continuity of dense bodies over considerable cell depth and their change in shape may have important implications for integration of contractile activity and for transmitting passive tension to the extracellular matrix.     

Some properties of the smooth muscle of rabbit portal vein

1. The morphology of the smooth muscle of the rabbit portal vein and its innervation were studied with fluorescence and electron microscopy. Two layers of smooth muscle were observed in the tunica media: an inner layer of circularly arranged muscle cells and an outer layer consisting of bundles of smooth muscle cells arranged in a near longitudinal direction. The membranes of neighbouring smooth muscle cells were occasionally fused to form ;tight junctions'.2. Bundles of non-myelinated nerve fibres were observed in the adventitia, and between bundles and layers of smooth muscle cells in the media. Studies on longitudinal sections with fluorescence microscopy revealed a network of varicose noradrenergic axons.3. Electrical and mechanical activity was recorded from longitudinal strips of smooth muscle from the media of the vein with a sucrose-gap apparatus.4. The preparation was spontaneously active under minimal resting tension (less than 150 mg) and at temperatures above 28 degrees C. Slow depolarizations led to a burst of spikes (multi-spike complexes), which corresponded to rhythmic contractions. In 10% of preparations, the interval between multi-spike complexes showed a slower depolarization, suggesting the record was from a pace-maker region.5. The frequency of spontaneous activity (3-27 beats/min) was very sensitive to changes in temperature and tension.6. Noradrenaline in low doses (0.01 mug) caused an increase in frequency of the multi-spike complexes. Higher doses (0.1-0.3 mug) initiated continuous high-frequency spiking, while very high doses (0.6-2.0 mug) caused maintained depolarization.7. Responses to repetitive electrical stimulation of the vein were qualitatively similar to those in response to exogenous noradrenaline. The relation between the mechanical response and the various parameters of stimulation was consistent with the stimulation of sympathetic nerve fibres in the wall of the vein.8. The actions of isoprenaline, phentolamine and propranolol indicated the presence of alpha ;excitatory' and beta ;inhibitory' adrenotrophic receptors on the smooth muscle.     

Lack of soluble carbonic anhydrase in aortic smooth muscle of the rabbit

Rabbit aorta was investigated for the occurrence of soluble, methazolamide-sensitive carbonic anhydrase (CA) by measuring electrometrically the rate of acidification of a weakly alkaline CO₂ solution buffered with 12.5 mM veronal/HCl at 0°C. For this purpose, aortae of 10 rabbits with the endothelium carefully preserved, were homogenized, centrifuged, and the supernatants pooled. The proteins were fractionated by FPLC, and tested for their CA activity. In control experiments, the pH change resulting from the spontaneous CO₂ hydration was found to be –0.92±0.01 pH units/min (mean±SE,n=10). Aliquots of the 30,000 dalton protein fraction corresponding to 100 mg of aortic tissue wet weight did not alter the hydration rate significantly (–0.94±0.01 pH units/min,n=10). Also, in the presence of 10^–4 M of the CA inhibitor, methazolamide, these rates were not altered significantly (–0.94±0.01 and –0.93±0.01 pH units/min, respectively,n=10). No CA activity was found in the other FPLC fractions, either. — These results suggest that soluble CA is absent from the myocytes and the endothelium of the rabbit aorta.Supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Three-dimensional structure of dense bodies in rabbit renal artery smooth muscle.

In this report, we present a three-dimensional computer assisted reconstruction study from serial thin sections through a rabbit renal artery smooth muscle cell. In a series of 32 consecutive thin (100-nm) sections, one longitudinally oriented cell was followed and photographed in alternating sections. The profiles of the cell surface and dense bodies were reconstructed from these 16 planes and the distribution, size, shape, and spatial relationships among these components was examined. The reconstructed images showed that the cell decreases in diameter from its widest region in the center to the two ends in a step-wise taper. Within the cell, dense bodies are numerous. Relative to the cell axes, a membrane associated dense body (MDB) can be less than or equal to 3.5 microns long, 0.25 micron wide, and may extend up to 2 microns in depth. While the MDB profile in one section may be aligned with the long axis of the cell, in an adjacent section the same dense body may appear almost circular or wedge shaped. The same is true of cytoplasmic dense bodies (CDBs). Compared with MDBs, CDBs are smaller in all dimensions. Some, but not all, CDBs line up in strings oriented with the long axis of the cell. The continuity of dense bodies over considerable cell depth and their change in shape may have important implications for integration of contractile activity and for transmitting passive tension to the extracellular matrix.     

Plexiform tumorlet of the uterus: immunohistological evidence for a smooth muscle origin

A case of plexiform tumorlet of the uterus is reported and studied histologically and by immunohistochemistry. The presence of desmin in the tumour cells demonstrates their smooth muscle origin. These results are compared with other reports in the literature with special emphasis on histogenesis.     

Potassium conductance of smooth muscle cells from rabbit aorta in primary culture

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1–7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of –50±0.3 mV (n=387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6±0.3 mV (n=45) and 16±2 mV (n= 5) when the bath K⁺ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba²⁺ (0.1–1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K⁺ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (g _K) of 209.6±4.6 mV (n=17) was read from the current/voltage curves at a clamp voltage (V _c) of 0 mV. After excision K⁺ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean g _K at a V _c of 0 mV was 134.6±3.9 pS (n=179). The mean permeability was 0.89±0.03×10^–12 cm³/s. With symmetrical KCl solution the mean g _K was 227±6 pS (n=17). The conductance sequence was g _K g _Rb= g _Cs=g _Na=0. TEA blocked dose-dependently only from the outside.(1–10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01–1 mmol/l) and quinine (0.01–1 mmol/l) blocked from both sides. Charybdotoxin (0.5–5 nmol/l) blocked only from the extracellular side. Ba²⁺ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a V _c of 0 mV a half-maximal inhibition (IC₅₀) of 2 mol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC₅₀ of 2 mol/l and diltiazem with an IC₅₀ of 10 mol/l. The open probability of this channel was increased by Ca²⁺ on the cytosolic side at activities > 0.1 mol/l. Half-maximal activation occurred at Ca²⁺ activities exceeding 1 mol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K⁺ conductance. In excised patches only a maxi K⁺ channel was detected. This channel has properties different from the macroscopic K⁺ conductance. Hence, it is likely that the K⁺ conductance of the intact cell is dominated by yet another and thus far not detected K⁺ channel.Supported by DFG Gr 480/10