Phosphatidylinositol-cleaving activity in smooth muscle from rat vas deferens

• 1.1. Phosphatidylinositol-cleaving activity was studied in subcellular fractions from smooth muscle of rat vas deferens.
• 2.2. In the presence of calcium ions and deoxycholate most of the endogenous phosphatidylinositol was broken-down in 60 min, whilst the other phospholipids were stable.
• 3.3. The enzymatic activity responsible for this breakdown catalyses a phospholipase C-type cleavage of the glycerol-phosphate bond, the water soluble products from exogenous [³²P]-labelled phosphatidylinositol being d-myoinositol 1:2-cyclic phosphate (702-80%) and d-myoinositol 1-phosphate (202-30%).
• 4.4. Activity was abolished by 1 mM ethanedioxybis(ethylamine)tetra-acetate (EGTA) and in the presence of deoxycholate both the soluble and total particulate fractions showed maximum activity at pH 6.52-6.8. The soluble fraction showed a second peak of activity at pH 5.52-5.8 that was independent of deoxycholate; this was not observed in the particulate fraction.
• 5.5. About two-thirds of the activity was soluble. The remaining activity was particulate, with a preferential concentration in the microsomal fraction.

     

Terminal axons ensheathed in smooth muscle cells of the vas deferens

Summary The ultrastructure of axon profiles which were completely ensheathed in smooth muscle cells has been described in the guinea pig, mouse and rat vas deferens. The axon profiles contained both small (500 Å) and large (1,000 Å) vesicles, neurotubules and mitochondria. Adrenergic axons were clearly identified within smooth muscle cells after treatment of the tissue with 5-or 6-hydroxydopamine, drugs which cause specific ultrastructural changes in adrenergic axons. The ensheathed axons were separated from the surrounding muscle cells by narrow, regular gaps, usually about 100–300 Å wide. Schwann cells seldom accompanied the ensheathed axons. Axons often penetrated the muscle cells in the nuclear region and profiles were sometimes observed among the perinuclear organelles.     

Involvement of calcium in calcium-current inactivation in smooth muscle cells from rat vas deferens

Summary Ca-channel currents were recorded in Cs-loaded single smooth muscle cells from rat vas deferens to define the dependence of the inactivation time course on Ca concentration. The decay of Ca-channel current obtained in a Ba²⁺- or Sr²⁺-containing external solution during long voltage-clamp pulses was much slower than that in a Ca-containing solution. The difference was not due to a change in the surface potential of the membrane as judged from the steady-state activation and inactivation curves. When Ca was the charge carrier, increasing external Ca concentration slightly accelerated the rate of inactivation. In addition, the rate of inactivation of Ca-channel current in 10.8mm Ba was also accelerated by adding Ca to the external solution in a concentration-dependent manner. The time course of Ca-current inactivation was slowed when the cells were dialyzed with a high concentration of citrate, a Ca-chelating agent. From these results, we concluded that a mechanism regulated by intracellular Ca activity plays a role in the inactivation of Ca channels in smooth muscle. The Ca-dependent process may protect against Ca overload by regulating Ca entry in smooth muscle cells.     

Characterization of outward K(+) currents in isolated smooth muscle cells from sheep urethra

The perforated-patch techniquewas used to measure membrane currents in smooth muscle cells from sheepurethra. Depolarizing pulses evoked large transient outward currentsand several components of sustained current. The transient current anda component of sustained current were blocked by iberiotoxin, penitremA, and nifedipine but were unaffected by apamin or 4-aminopyridine,suggesting that they were mediated by large-conductanceCa²⁺-activated K⁺ (BK) channels. When the BKcurrent was blocked by exposure to penitrem A (100 nM) andCa²⁺-free bath solution, there remained a voltage-sensitiveK⁺ current that was moderately sensitive to blockade withtetraethylammonium (TEA; half-maximal effective dose = 3.0 ± 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increasedthe spike amplitude and plateau potential in slow waves evoked insingle cells, whereas addition of TEA (10 mM) further increased theplateau potential and duration. In conclusion, bothCa²⁺-activated and voltage-dependent K⁺currents were found in urethral myocytes. Both of these currents arecapable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.

     

TTX-sensitive Na(+) and nifedipine-sensitive Ca(2+) channels in rat vas deferens smooth muscle cells.

The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.     

Reinnervation of regenerating smooth muscle cells in minced vas deferens of the guinea-pig

Summary In adult guinea-pigs, a portion of the wall of the vas deferens was removed, minced and replaced. This caused muscle cells to dedifferentiate, divide and redifferentiate. Reinnervation of redifferentiating cells was followed using electron microscopy and histochemistry. Adrenergic nerves were first observed to re-enter the regenerating area 5 days after operation, and close contacts (within 20 nm) with muscle cells were first seen at 10 days. The total number of adrenergic nerves per 100 muscle cells reached control values by 5 weeks, and by 15 weeks was higher than control levels. Cholinergic nerves first appeared in the regenerating area about 3–4 weeks after the operation. The total number of cholinergic nerves present had not reached control values even at 15 weeks, and no nerve muscle contacts within 20 nm were observed. The ratio of adrenergic to cholinergic nerves in the regenerating area was higher at 15 weeks than in control tissue.This work was supported by grants from the Wellcome Trust and the Medial Research Council     

Electrophysiological recordings from spontaneously contracting reaggregates of cultured smooth muscle cells from guinea pig vas deferens

Smooth muscle cells were enzymatically dispersed from vasa deferentia of adult male guinea pigs (250-400 g). These cells reassociated in vitro to form monolayers and small spherical reaggregates (0.05-0.3 mm in Diam). Within 48 h of being placed in culture, cells in both types of preparation began to contract spontaneously. The contractions were rhythmic and slow. Cells in the monolayers stopped contracting after approximately 1 wk in vitro, but the reaggregates continued to contract spontaneously for at least 3 wk. Electron microscopy of the reaggregates revealed the presence of thick and thin myofilaments. Overshooting action potentials were recorded in many of the cells penetrated (primarily in reaggregates), and were accompanied by visible contractions of the aggregate or monolayer. Quiescent cells could often be excited by intracellularly applied depolarizing and hyperpolarizing (anodal-break) current pulses. The resting potentials had a mean value of -58 +/- 2 mV. The action potentials were usually preceded by a spontaneous depolarization. The action potentials had slow rates of rise (1--4 V/s) which were unaffected by tetrodotoxin (TTX, 1 microgram/ml), a known blocker of fast Na+ -channels. Verapamil (1 microgram/ml) blocked the action potentials. The mean value of input resistance was 6.9 +/- 0.5 M omega (n = 12). These electrophysiological properties are similar to those of intact adult vas deferens smooth muscle cells. Thus, the cultured adult vas deferens smooth muscle cells retain their functional properties in vitro even after long periods.     

Calcium channel currents in isolated smooth muscle cells from human bronchus

An electrophysiological study was carried out on smooth muscle cells that were enzymatically dissociated from bundles of muscle fibers dissected out of human bronchi obtained at thoracotomy. These cells that retain the contractile properties of intact bundles were voltage-clamped by means of the whole-cell patch-clamp technique. Upon voltage steps from a holding potential of -60 mV to more positive levels, the initial inward current was followed by large outward currents that inactivated slowly. These were subsequently reduced by substituting Cs+ for K+ in the internal solution and by using Ba2+ instead of Ca2+ as a charge carrier in the external solution. Under these conditions, the inward current did not completely inactivate in the course of 300-ms voltage steps. Inward current measured after leak subtraction was activated at a membrane potential of -25.8 +/- 5 mV, was maximum at +18 +/- 4 mV, and had an apparent reversal potential of +52.5 +/- 5.5 mV (n = 5). The potential at which steady-state inactivation was half-maximum was -28 mV (n = 5). This inward current was identified as a calcium current on the following basis: 1) it was not altered by 10 microM tetrodotoxin (TTX) or by lowering to 10 mM external Na+ concentration; 2) it was blocked by 2.5 mM Co2+ or 1 microM PN 200-110; 3) it was enhanced by 1 microM BAY K 8644, which in addition suppressed the PN 200-110 blockade.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis

The ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis were examined by using conventional two-electrode voltage clamp and whole-cell patch clamping methods. Several separable currents were identified. These include: (1) a transient and (2) a steady-state voltage-activated inward current; both are tetrodotoxin (TTX) and saxitoxin (STX) insensitive, partly reduced by decreasing external Ca2+ or Na+ or by addition of 5 mM Co2+, D-600 or verapamil and are totally blocked with 5 mM Cd2+; (3) an early, transient, cation-dependent, outward K+ current (IKCa/Na); (4) a transient, voltage-activated, outward K+ current provisionally identified as IA; (5) a delayed, steady-state, voltage-activated outward K+ current (IK) and (6) a late, transient, outward K+ current which is blocked by Cd2+ and evident only during long voltage pulses. Despite their phylogenic origin, most of these currents are similar to currents identified in many vertebrate smooth and cardiac muscle preparations, and other excitable cells in higher animals.     

Dense-cored membranous structures in smooth muscle cells of the vas deferens of the rat

Summary Smooth muscle cells from rat vas deferens were studied by electron microscopy. Vesicular and tubular membranous structures containing an electron-opaque material were found in the smooth muscle cells. Similar structures were also found in a subfraction (F3) of microsomes of vas deferens smooth muscle which was shown to be rich in both plasma membrane and putative endoplasmic reticulum markers. Treatment of the tissues with calcium-free Krebs solution containing EGTA prior to fixation eliminated almost completely the presence of these dense-cored membranous structures (DMS), whereas incubation of the subcellular membrane fraction with EGTA solution had no effect on the appearance of the DMS. Plasma membrane infoldings were found in the smooth muscle cells extending well into their interior. Horseradish peroxidase penetrates vesicles in a location similar to that of DMS in smooth muscle cells, suggesting that some of the DMS may be connected to the extracellular space. We conclude that the dense-core material within the DMS is calcium dependent. We also suggest that some of the DMS represent infoldings of the plasma membrane extending into the cell's interior.     

The nervous environment of individual smooth muscle cells of the guinea pig vas deferens

Smooth muscle cells of the external longitudinal coat of the guinea pig vas deferens were followed for 480 mu at 4.5-mu intervals. Muscle bundles and fibers interwove, facilitating intermuscular and neuromuscular contacts. The ribbon- or rodlike muscle cells were about 450 mu long, 3,000 mu3 in volume, and 4,500 mu2 in area. The thickened nuclear zone day anywhere along the middle one-third of the cell. Intercellular distances were 500-800 A. Intrusions were rare, and tight-junctions absent. At any level in a field of 80 muscle fibers there were 10-15 nerve bundles, each containing several varicose axons. Bundles and axons divided. Axons, en passage, were frequently within 500-1,000 A of a muscle fiber. En passage close contacts were rate. Axon terminations were bare, and bare axons invariably terminated. Bare terminations had scattered vesicle-laden varicosities and were from 10-60 mu in length, and all ended within 500 A of muscle fibers. Some made close contact with muscle fibers. Less than half of the muscle cells received this close contact, but some cells were approached by more than one termination. Most terminations involved more than one cell. Some cells had little or no innervation. Some groups of cells had a rich innervation. There was very little evidence of sensory innervation. These conclusions are not valid for other smooth muscles.     

Facilitated diffusion of monosaccharides in smooth muscle of rat vas deferens in vitro

The suitability of rat vas deferens for investigating sugar transport in smooth muscle was determined in vitro, with the nonmetabolized glucose analog 3-O-methyl-D-glucose as test sugar. Vas deferens smooth muscle contains a facilitated diffusion system for monosaccharides, as shown by saturation of the transport sites and by competition between 3-O-methyl-D-glucose and D-glucose. The activity of the facilitated diffusion system could be enhanced by hyperosmolarity and by contractile activity, but frequency dependency could not be established. A high concentration of insulin (100 mU/mL) was required to stimulate sugar transport. As smooth muscle is not a primary tissue for the storage of energy reserves, it does not require large numbers of insulin receptors.