Spatial relationships between sympathetic varicosities and smooth muscle cells in the longitudinal layer of the mouse vas deferens

Summary The spatial relationships between nerve varicosities and smooth muscle cells in the longitudinal muscle layer of the mouse vas deferens have been determined from serial section reconstructions of individual varicosities at the ultrastructural level. Bundles of up to five axons, together with single axons, occurred frequently at the surface of the muscle as well as at about 3–6 muscle cell diameters into the muscle. Varicosities within axon bundles at the muscle surface each became partially divested of Schwann cell processes. The smallest distance separating varicosity membrane from muscle cell membrane (apposition distance) was 100 nm (mean 170 nm) for varicosities contained in bundles. Varicosities from six single axons on the muscle surface were reconstructed and 11 of the 12 possessed a mean apposition distance of 48 nm. Varicosities in axon bundles at about 12 m deep into the muscle came into an apposition distance of 50–90 nm (mean=67 nm). All varicosities of single axons at this depth came into about 50 nm apposition (mean=53nm). These results indicate that the varicosities lie at varying distances from the muscle cells in the longitudinal muscle layer of the vas deferens.     

Fast and slowly inactivating components of Ca-channel current and their sensitivities to nicardipine in isolated smooth muscle cells from rat vas deferens

(1) Fast and slowly inactivating components of Ca-channel current were compared to clarify whether more than one type of Ca-channel exists in smooth muscle cells from rat vas deferens using the whole cell variant of the patch clamp technique. The pipette was filled with 150 mM Cs solution to eliminate outward current and Ba was used as the charge carrier for Ca-channel current. (2) When activated by a 5 s test pulse to 0 mV from a holding potential of −60 mV, the inactivation process of Ba-current was well fitted by the sum of two exponentials. The time constant of the faster inactivating component was 100–300 ms and that of the slower inactivating component was 1.5–3 s. Steadystate inactivation curves of the fast- and slow-components were very similar. (3) The inward current activated at 0 mV from −80 mV was inactivated faster than that from −30 mV. The voltage-dependencies of the peak current from holding potentials of −30 mV and −80 mV were similar. Both had voltage threshold at −30 mV and were maximal at +10 mV. (4) Low concentrations of nicardipine (10⁻⁹ to 10⁻⁷ M) preferentially inhibited the slow component while higher concentration (10⁻⁶ to 10⁻⁵ M) were required to block the fast component. The current activated from a holding potential of −30 mV was almost fully suppressed by 10⁻⁷ M nicardipine whereas that from −80 mV was blocked only slightly. The voltage dependencies of the peak currents before and during the superfusion with nicardipine (10⁻⁷ M) were similar although the peak amplitude was suppressed in the presence of the drug. (5) These results suggest that the existence of either (a) two populations of Ca channels that differ in the time course of inactivation and the sensitivity to nicardipine, but have nearly identical dependence on membrane potential or (b) one population of Ca channel having two different states of inactivation and the sensitivity of nicardipine, in rat vas deferens.     

Current spread in the smooth muscle of the guinea-pig vas deferens

1. The membrane polarization in response to intracellular stimulation and external stimulation, the junction potentials evoked by nerve and field stimulation and the spontaneous junction potentials were studied in the guinea-pig vas deferens.

2. The responses to intracellular stimulation differed from those to external stimulation applied through a large electrode in the following ways: short time constant of the electrotonic potential; linearity of current—voltage relation; all-or-none spike only in a small proportion of the cells; high critical firing level; short latency; weak tendency for repetitive activity during depolarization; and sharp spatial decay of the response.

3. The difference between intracellular and external stimulation could be explained by differences in current distribution in the tissue, if many muscle fibres were aggregated in functional bundles, with three-dimensional cell-to-cell connexions, so that the membrane near an intracellular stimulating electrode was shunted by a large area of surrounding membrane.

4. The time course of the junction potentials depended on the manner by which they were produced. The junction potential evoked by hypogastric nerve stimulation was recorded in every cell with almost the same amplitude. The spontaneous junction potential decayed very sharply with distance and the time course of the falling phase was about 10 times faster than that of the evoked junction potential.

The difference between the time course of the junction potentials was also explained by the difference in current distribution in the tissue.

     

Adenosine triphosphate-activated inward current in isolated smooth muscle cells from rat vas deferens

The electrophysiological effect of adenosine triphosphate (ATP) on the enzymatically dispersed smooth muscle cells from rat vas deferens was investigated. ATP always induced depolarization accompanied with a reduction in membrane resistance. In a whole cell voltage clamp experiment, an inward current was recorded when the cell was exposed to ATP-containing solution. The ATP-induced current disappeared within 2min even in the continuous presence of ATP, which may indicate that the cells were desensitized to this compound. The ATP-induced current was also recorded in the cells superfused with 10^–5M nicardipine or in the Cs-loaded cells, eliminating the possible involvement of voltage-gated Ca and K current. During cell-attached patch clamp, an elementary current having a mean conductance of 20pS was observed when the intrapipette solution was changed to ATP-containing solution. The estimated zero current potentials of the ATP-activated macroscopic current and elementary current were about 0mV. These results suggest that ATP exerts its transmitter-like action by activating ion channels in smooth muscles.     

Volume-sensitive chloride currents in primary cultures of human fetal vas deferens epithelial cells

Using the patch-clamp technique, we have identified a large, outwardly rectifying, Cl⁻-selective whole-cell current in primary cultures of human vas deferens epithelial cells. Whole-cell currents were time- and voltage-dependent and displayed inactivation following depolarising pulses ≥ 60 mV. Currents were equally permeable to bromide (P _Br/P _Cl = 1.05 ± 0.04), iodide (P _I/P _Cl = 1.06 ± 0.07) and Cl⁻, but significantly less permeable to gluconate (P _Gluc/P _Cl = 0.23 ± 0.03). Currents spontaneously increased with time after establishing a whole-cell recording, but could be inhibited by exposure to a hypertonic bath solution which reduced inward currents by 68 ± 4%. Subsequent exposure of the cells to a hypotonic bath solution led to a 418 ± 110% increase in inward current, indicating that these currents are regulated by osmolarity. 4,4′-Diisothiocyanatostilbene-2,2′-disulphonic acid (100 μM) produced a rapid and reversible voltage-dependent block (60 ± 5% and 10 ± 7% inhibition of current, measured at ± 60 mV, respectively). Dideoxyforskolin (50 μM) also reduced the volume-sensitive Cl⁻ current, but with a much slower time course, by 41 ± 13% and 32 ± 16% (measured at ± 60 mV, respectively). Tamoxifen (10 μM) had no effect on the whole-cell Cl⁻ current. These results suggest that vas deferens epithelial cells possess a volume-sensitive Cl⁻ conductance which has biophysical and pharmacological properties broadly similar to volume-sensitive Cl⁻ currents previously described in a variety of cell types. Received: 25 January/Accepted: 25 April 1996     

Contractile response and electrophysiological properties in enzymatically dispersed smooth muscle cells of rat vas deferens

Electrophysiological studies were performed on single smooth muscle cells isolated from the vas deferens of the rat. The tissue was preincubated in Ca-free modified Tyrode's solution for 1 h and then transferred to a high-K solution for 1 h. It was next minced and treated with the enzyme solution composed of 600–800 unit/ml collagenase and 40 unit/ml elastase. The procedure yielded about 50% spindle shaped Ca-tolerant cells (100–250 m in length and about 10 m in diameter). These cells could contract during the superfusion with the solutions containing 10^–8 to 10^–3M norepinephrine (NE) or adenosine triphosphate (ATP). The cells isolated from the epididymal portion were more sensitive to norepinephrine than were those from the prostatic part. Their basic electrical properties were studied using tight-seal suction electrode technique. The cells had resting potentials around –40 mV and their input resistance was about 0.8 G. Action potentials could be evoked by application of depolarizing current. During whole cell voltage clamp, an inward current followed by an outward current was recorded when 800 ms pulses from a holding potential of –60 mV to test potentials positive than –40 mV were applied. The transient outward current generally recorded in other smooth muscle cells was not seen in these cells. The amplitude of the inward current was Ca dependent and sensitive to a Ca antagonist, nicardipine, indicating that Ca ion is the main carrier of this component of the current. When the pipette was filled with Cs-containing solution, the outward current was abolished. In this condition, the reversal potential of Ca current was +53.4 mV, and the time course of inactivation was composed of more than one exponential component. The results suggest that these isolated cells retain many characteristics of analogous multicellular preparation and that they are a useful model of the postsynaptic properties in smooth muscle especially when studied electro-physiologically.     

Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse

We compared the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), as measured with the fluorescent Ca²⁺ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K²⁺ depolarization and by agonists. Resting [Ca²⁺]_i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca²⁺ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca²⁺]_i increase, the agonist stimulation induced a higher force development than the K⁺ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca²⁺]_o. Our results indicate that the Ca²⁺ threshold for force development was about 150% of the resting [Ca²⁺]_i in both cell types. The resting [Ca²⁺]_i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca²⁺ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca²⁺ sensitivity during contraction. The desensitization to Ca²⁺ was specific for phasic muscle, in which it occurred as an early, timeand Ca²⁺-dependent process that was partially reversible.     

The effect of cations on the electrical properties of the smooth muscle cells of the guinea-pig vas deferens

1. A study has been made of the effects of Na(+) and Ca(2+) on the responses generated by intracellular current pulses in smooth muscle cells of the guinea-pig vas deferens and upon the propagated action potential.2. Reduction of the extracellular Na(+) activity (a(Na)) to less than 30 mM did not alter the characteristics of the spike-like response to intracellular current pulses (the active response) or of the propagated action potential.3. Reduction of the extracellular Ca(2+) activity (a(Ca)) to 0.1 mM decreased the resting input resistance of the cells but increased the input resistance during the crest of the active response. Reduction of Ca(2+) decreased the overshoot of the action potential by 22 mV per tenfold change in Ca(2+) and the resting potential by 25 mV per tenfold change.4. Reduction of the extracellular Ca(2+), keeping the product a(Ca)/a(2) (Na) constant, did not change the resting potential, but decreased the action potential overshoot by 20 mV per tenfold change.5. It is suggested that part of the current responsible for the rising phase of the action potential is carried by Ca(2+).     

Peristaltic transport of a non-newtonian fluid: Applications to the vas deferens and small intestine

The problem of peristaltic transport of a non-Newtonian (Power law) fluid in a uniform and non-uniform tube has been investigated under zero Reynolds number and long wavelength approximation. A comparison of the results with those of Newtonian fluid model shows that the magnitude of pressure rise, under a given set of conditions, is smaller in the case of non-Newtonian fluid, when the flow behavior index n<1, at zero flow rate. Further, the pressure rise decreases as n decreases from 1, at zero flow rate, is independent of n at a certain value of flow rate, and increases if flow rate exceeds further. Also, at a given flow rate, an increase in the wavelength leads to a decrease in pressure rise and increase in the influence of non-Newtonian behavior. Pressure rise, in the case of non-uniform geometry, is found much smaller than the corresponding value in the case of uniform geometry. Finally, the analysis has been applied and compared with observed flow rates in the vas deferens in rhesus monkeys and in the small intestine.     

Decrease in membrane conductance induced by noradrenaline in the smooth muscle of guinea-pig vas deferens

The electrical response of the smooth muscle of guinea-pig vas deferens to exogenously applied noradrenaline (NA) was examined using the double sucrose-gap method. NA evoked a depolarization of the smooth muscle membrane which was associated with an increase in the size of electrotonic potentials. A conditioning depolarization of the membrane induced by current application enhanced the size of NA-induced depolarization, whereas a conditioning hyperpolarization reduced it. When a conditioning hyperpolarization of 25 mV in magnitude was applied, the direction of potential change induced by NA was reversed. These results are discussed with respect to the ionic mechanism of the electrical event in response to NA in this tissue.     

Dual excitatory actions of acetylcholine at the neuromuscular junction in the guinea-pig vas deferens

The action of acetylcholine (ACh) on the smooth muscle of guinea-pig vas deferens was studied using the sucrose-gap method. ACh, when applied at a concentration of 10^–6 M, evoked a depolarization of the smooth muscle membrane which was slow in time course (slow depolarization). When ACh was applied at higher concentrations, another depolarization which was fast in time course (fast depolarization) occurred, overlapping the early part of the slow depolarization. The magnitudes of both depolarizations were concentration-dependent on ACh. TTX and adrenergic receptor antagonists had little effect on either depolarizations, while guanethidine and nicotinic receptor antagonists mainly suppressed the fast depolarization. In contrast, atropine suppressed the slow depolarization. The membrane conductance observed by current application, was reduced during the slow depolarization, and the reversal potential of the depolarization was 18.3 mV negative to the resting membrane potential. Whereas, the reversal potential of the fast depolarization was 27.6 mV positive to the resting membrane potential. This reversal potential was quite similar to that of the adenosine triphosphate (ATP)-induced depolarization, previously observed in the same tissue. From these observations, it is suggested that in the guinea-pig vas deferens, ACh acts on nicotinic receptors at the sympathetic postganglionic nerve terminal, causing the release mostly of a non-adrenergic transmitter, probably ATP. In addition, ACh also acts on muscarinic receptors on the smooth muscle membrane, inducing membrane depolarization resulting from a reduction of the membrane conductance to potassium ions.     

The distribution of chloride ions in the smooth muscle cells of the guinea-pig's taenia coli

1. The intracellular Cl concentration of taenia coli cells, determined by an analytical procedure and by an extrapolation procedure, has a value between 60 and 73 m-mole/l. cell water.2. This concentration is too high to be explained by a passive distribution. The discrepancy could be due to a binding of Cl in the intracellular or extracellular compartment or to an active uptake of Cl by the cells.3. Determination of the activity coefficient for Cl in homogenates of smooth muscle did not support the hypothesis of binding of Cl ions.4. The efflux of (36)Cl from taenia coli cells was not affected by foreign anions. After 1 hr exposure to a Cl-free solution, the tissues contained less than 1 m-mole of Cl/kg wet wt., even if Cl had been replaced by a slowly penetrating anion. Because the intracellular cation concentration remained constant, it has to be assumed that new anionic groups can be formed in the cells.5. The intracellular Cl concentration decreases during exposure to ouabain or to K-free solution. The uptake seems therefore to be linked to the uptake of K through the Na pump.6. Exposure to K-free solution increases the K permeability of the membrane. Under the same experimental conditions the Cl permeability of the membrane increases as long as K is leaking out of the cells.7. The anions in the external solution exert an important influence on the K permeability of the membrane. NO(3) and I cause a small increase of the permeability and large anions such as benzenesulphonate, propionate or pyroglutamate cause a pronounced decrease of this permeability.     

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...     

Postnatal androgen deprivation dissociates the development of smooth muscle innervation from functional neurotransmission in mouse vas deferens

The pelvic autonomic nervous system is a target for circulating androgens in adults, with androgen exposure or deprivation affecting the structure and function of urogenital tract innervation. However, the critical period for androgen exposure to initially establish pelvic autonomic neuromuscular transmission has not been determined. We have examined the sympathetic innervation of the vas deferens in hypogonadal ( hpg ) mice that are deprived of androgens after birth but undergo normal prenatal sexual differentiation and remain androgen responsive throughout life. In vasa deferentia from hpg mice, purinergic excitatory junction potentials and contractions could not be elicited by electrical stimulation and P2X₁ purinoceptors could not be demonstrated by immunofluorescence. Moreover, a novel inhibitory nitrergic transmission developed. Administering testosterone to adult hpg mice restored purinergic excitatory transmission and P2X₁ purinoceptor immunofluorescence, and nitrergic inhibitory transmission was lost. Despite the deficit in excitatory neurotransmission in hpg mice, their vasa deferentia were innervated by numerous noradrenergic axons and pelvic ganglia appeared normal. In addition, noradrenergic contractions could be elicited by electrical stimulation. This study has revealed that postnatal androgen exposure has a profound effect on the development of excitatory transmission in vas deferens smooth muscle, primarily by a postjunctional action, but is not essential for development of the structural innervation of this organ. Our results also indicate that there is no postnatal critical period for androgen exposure to establish neuroeffector transmission and that postnatal androgen exposure can be delayed until adulthood, with little consequence for establishment of normal sympathetic neurotransmission.     

A comparison of the electrical properties and morphological characteristics of the smooth muscle of the rat and guinea-pig vas deferens

Summary Microelectrodes were used to compare a variety of electrophysiological parameters of the rat and guinea-pig vas deferens. In comparison to the guinea pig, spontaneous junction potentials in the rat tissue were of shorter duration and occurred with greater frequency and amplitude. Action potentials induced by nerve stimulation could be observed in the smooth muscle of both species. However, in the rat tissue the majority of action potentials were generated in the impaled cell while 60% of the action potentials in the guinea-pig vas deferens were propagated. When current was intracellularly applied, spike potentials could be induced in approximately 90% of the cells of the rat vas deferens but in less than 10% of the cells of the guinea-pig vas deferens, The space constant was 1.48 mm for the guinea-pig vas deferens, but less than 0.5 mm for the rat vas deferens. Electronmicroscopic examination of the homologous tissues indicates that the differences in electrical properties can be accounted for in part by differences in morphology. The incidence and intimacy of neuromuscular contacts was greater in the rat vas deferens while the incidence of nexuses between smooth muscle cells was greater in the guinea-pig tissue.Supported by grants from the National Institute of Neurological Diseases and Stroke (NS 08300) and the West Virginia University Medical Corporation     

Single anion-selective channel and its ion selectivity in the vascular smooth muscle cell

The properties of voltage-gated Cl channels of cultured smooth muscle cell prepared from embryonic rat aorta were studied. In the excised patch (inside-out configuration), we observed the activity of channels, opening and closing spontaneously, when the membrane potential was held at around 0 mV. The channels were active at a potential range between +10 and ?10 mV. A step change of the membrane potential from the active potential range in either a positive or a negative direction closed the channel to an apparently inactivated state. The time course of this inactivation process became faster as the amplitude of the step change was increased. Returning the membrane potential to 0 mV allowed the channel to recover from the inactivated state. The channel had at least two open conductance states. Ca ions at the cytoplasmic face were not required for the activation of the channel. Adenosine nucleotides at the same side of the membrane had no effect on channel activity. The channels were selective to anions rather than cations, and they had a large single channel conductance of 340.5±20.4 pS in symmetrical 150 mM TEA-Cl. The reversal potential of the channel was shifted by ?15.2±2.6 and 17.0±1.7 mV, when the Cl concentration at the intracellular side was changed to 75 mM or 300 mM, respectively. The permeability sequence of halides was I^?>Br^?>Cl^?>F^? (1.4∶1.3∶1.0∶0.7), whereas the conductance sequence was Cl^?>Br^?>F^?>I^? (1.00∶0.89∶0.86∶0.83). The internal dimension of the channel was estimated by measuring the permeability of various anions with different molecular cross section. We suggest that the smallest cross section of the channel pore is about 32Ų.     

86Rb+ ion fluxes in resting and immunologically stimulated mucosal mast cells

We studied fluxes of Rb⁺ ions, using its ⁸⁶Rb isotope as a radioactive tracer in living rat mucosal mast cell cultures (RBL-2H3 line) grown to high density on beads. Continuously perfused samples of these cells could be immunologically stimulated by antigen clustering of IgE bound to the cells type I Fee receptors (FcεRI) and both the cellular response, as measured by the secreted mediators, as well as the uptake of ⁸⁶Rb⁺ of the perfused sample could be monitored. The following results were obtained, (i) In resting cells, ⁸⁶Rb⁺ influx is observed upon exposure to extracellular ⁸⁶Rb⁺. It proceeds with a monoexponential time course (τ = 30.6 ± 8 min) reaching a steady-state distribution of [⁸⁶Rb⁺]^int/[⁸⁶Rb⁺]^ext ₌ 31.6 ± 6.4 and can be inhibited by ouabain, (ii) FcεRI clustering-mediated stimulation of these cells causes an immediate and marked increase in both amplitude and rate of ⁸⁶Rb⁺ uptake, which also fits a monoexponential function (τ = 26.8 ± 8.6 min). (iii) This stimulated ⁸⁶Rb⁺ uptake can also be inhibited by ouabain. It is not caused by Ca²⁺ influx or by the exocytotic process as evidenced by the fact that it is also observed in buffer to which no Ca²⁺ ions were added. Analysis of these results by a simple model taking into account unidirectional ⁸⁶Rb⁺ influx by the Na⁺/K⁺-dependent ATPase and its efflux by K⁺ channels yields a resting cells unidirectionel K⁺ uptake of 3.0 ± 1.1 10⁷ ions/cell/s, which is increased by ca. 10% upon clustering of the FcεRI by IgE and antigen. The stimulated influx is suggested to be due to enhanced activity of the Na⁺/K⁺-dependent ATPase, reflecting increased permeability for Na⁺ ions.     

Ion channels and ion transporters of the transverse tubular system of skeletal muscle

This review focuses on the electrical properties of the transverse (T) tubular membrane of skeletal muscle, with reference to the contribution of the T-tubular system (TTS) to the surface action potential, the radial spread of excitation and its role in excitation-contraction coupling. Particularly, the most important ion channels and ion transporters that enable proper depolarization and repolarization of the T-tubular membrane are described. Since propagation of excitation along the TTS into the depth of the fibers is a delicate balance between excitatory and inhibitory currents, the composition of channels and transporters is specific to the TTS and different from the surface membrane. The TTS normally enables the radial spread of excitation and the signal transfer to the sarcoplasmic reticulum to release calcium that activates the contractile apparatus. However, due to its structure, even slight shifts of ions may alter its volume, Nernstian potentials, ion permeabilities, and consequently T-tubular membrane potential and excitability.