Endocytic appartus and transcytosis in epithelial cells of the vas deferens in the rat

The apex of the principal epithelial cells lining the vas deferens of the rat contains coated pits in continuity with the apical plasma membrane and large subsurface-coated vesicles (100–125 um). In the apical cytoplasm, large, pale, uncoated vesicles (150–300 nm), small coated and uncoated vesicles (50–60 nm), uncoated vesicles about 75–90 nm, and membranous apical tubules are present, in addition to large, vacuolar, pale, multivesicular bodies, dense multivesicular bodies, and secondary lysosomes seen deeper in the cytoplasm amongst numerous ER cisternae, saccules of the Golgi apparatus, and mitochondria. The endocytic activity of these cells was investigated by using cationic ferritin (CF) as a marker of adsorptive endocytosis and native ferritin (NF) for demonstrating fluid-phase endocytosis. These tracers were injected separately into the lumen of the vas deferens, and the animals were killed at various time intervals thereafter from 2 to 90 minutes. At 2 minutes CF was seen bound predominantly to microvilli and to areas of the apical plasma membrane delimiting coated pits as well as in large, coated vesicles. At 5 and 15 minutes the tracers were seen in apical tubules and pale multivesicular bodies; at 30 minutes moderately dense multivesicular bodies were labeled. At 1 hour and longer time intervals dense multivesicular bodies and secondary lysosomes were labeled. NF followed the same pathway as CF; however, no binding to microvilli or areas delimiting coated pits was observed. The numerous other vesicular structures, i.e., the large uncoated vesicles (150–300 nm) and the small coated and uncoated vesicles (50–60 nm), never became labeled with the tracers and therefore were not involved in the endocytic process. There was, however, and exception in the case of several small (75–90 nm) uncoated vesicles seen deeper in the apical cytoplasm of these cells which were labeled exclusively with CF. With time such vesicles appeared along the lateral and basal surfaces of these cells and discharged their content of CF into the lateral intercellular space or the connective tissue space at the base of these cells. Thus the principal epithelial cells in addition to sequestering the endocytosed tracers within secondary lysosomes where they are presumably degraded also appear to be involved in the transcytosis of material from the lumen of the vas deferens to the underlying lamina propria.     

Voltage-gated potassium currents in rat vas deferens smooth muscle cells

Voltage-gated components of the outward current in single smooth muscle cells isolated from the epididymal part of the rat vas deferens were studied using amphotericin B perforated patch-clamp techniques. The complex kinetics of the net outward current elicited by positive voltage steps from -80 mV to +40 mV suggested the presence of several components. Bath application of 200 nM charybdotoxin, a potent blocker of large-conductance, Ca(2+)-dependent K(+) channels (BK(Ca)), reduced the current amplitude significantly. When BK(Ca) channels were suppressed, fast-inactivating (I(K,f)) and delayed rectifying (I(K,dr)) components of the outward current were identified. I(K,f) was characterized by fast kinetics of current decay, negative steady-state activation and inactivation dependencies and sensitivity to 4-aminopyridine with an apparent K(d) of 0.32 mM, properties similar to those of the A-type K(+) current. In contrast, I(K,dr) activated and inactivated at more positive potentials. The time constant of activation of I(K,dr) was voltage dependent with an e-fold decrease per 21 mV depolarization. I(K,dr) was inhibited by clofilium, a blocker of voltage-gated K(+) channels, with an IC(50) of 12 micro M and was not blocked by 5 mM 4-aminopyridine. The possible significance of the voltage-gated currents is discussed.     

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     

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.     

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.     

Effect of nonachlazine on adrenergic neurotransmission in the rat vas deferens

The effect of a new antianginal preparation, nonachlazine, on adrenergic neurotransmission in the isolated rat vas deferens was studied by recording contractions of the duct in response to transmural stimulation of postganglionic sympathetic nerves by the electric current or by application of noradrenalin (NA) or BaCl₂. The effect of nonachlazine on the NA content was studied spectrofluorometrically and the ability of the preparation to block the uptake of exogenous NA by the tissues also was investigated. Nonachlazine was found to have a moderate sympatholytic action, which was combined with its spasmolytic effect. Nonachlazine also has a well marked ability to block the reaccumulation of NA in the tissues.     

Gross and histological variations along the length of the rat vas deferens

The gross morphology of the vas deferens of the rat and the changes that occur along its length are reported. It is shown that the organ can be divided into proximal, distal and terminal portions. Each portion is histologically unique and is situated in a different part of the body. The differences are discussed in relation to the function of the organ and their possible role in the consequences of vasectomy.     

Sympathetic efferent pathways projecting bilaterally to the vas deferens in the rat

ABSTRACT Background: The laterality of the signals passing through the splanchnic nerves to the vas deferens has not been well studied. Methods: The present study was designed to determine the bilateral distribution of sympathetic nerves to the rat vasa deferentia by measuring intravasal pressure (VP) responses to electrical stimulation of left lumbar splanchnic nerves (LSN) following consecutive transections of more distal nerves. Results: L₂-L₆ LSN stimulation increased VP bilaterally. Left VP responses decreased slightly (<20%) after section of the right hypogastric nerve (HGN) and then were abolished by subsequent section of branches (B-M-APG) between the left major pelvic (MPG) and accessory pelvic ganglia (APG). Left VP responses were decreased by >80% after section of left HGN, not changed further by subsequent section of commissural branches (CB-MPG) between the MPG, and completely eliminated by section of commissural branches between the APG (CB-APG). Right VP responses were decreased slightly (<20%) by section of the left HGN and then abolished by section of the right B-M-APG. These responses were also decreased by >70% by section of right HGN, not changed by section of CB-MPG, but then completely eliminated by section of CB-APG. Conclusions: These results indicate that the left lumbar sympathetic pathway to the vas deferens is distributed bilaterally and exhibits two crossing points at the level of the inferior mesenteric ganglion and APG. Anat. Rec. 248:291-299, 1997. © 1997 Wiley-Liss, Inc.     

Electroimmunoblotting of neuropeptide Y: application to the rat vas deferens

In this study we have optimized the electroimmunoblotting conditions for neuropeptide Y (NPY). NPY standards and samples extracted from the rat vas deferens were separated on urea-sodium dodecyl sulphate gels. Densitometric scanning of the Coomassie Blue-stained gels allow a semi-quantitative analysis of NPY in the range of approximately 10(-11) to 10(-8) mol of NPY. Electroimmunoblotting of NPY was also shown to be best achieved overnight at 4 degrees C and with NC membranes of 0.22 micron. Under these conditions NPY extracted from the vas deferens has been efficiently electroimmunoblotted. Higher molecular weight NPY-reactive peptides were also detected that may be related to proteolytic processing of the NPY precursor.     

The mechanism of Ptychodiscus brevis toxin-induced rat vas deferens contraction

Ptychodiscus brevis, which causes Florida red tide, produces Ptychodiscus brevis toxin (PBTX) known to contain neurotoxins and to induce rhinorrhea, tearing, and cough in normal humans and wheezing in asthmatic subjects. It was previously reported (J Allergy Clin Immunol 69:418, 1982; 73:824, 1984) that PBTX causes canine tracheal smooth muscle contraction via stimulation of sodium channels in the axons of parasympathetic postganglionic nerves and the release of acetylcholine from these nerve endings. This was postulated to be an asthma-triggering mechanism. In this article the toxins were evaluated to determine if they also stimulate sodium channels on adrenergic nerve endings and release norepinephrine. Rat vas deferens was selected as the experimental tissue. Both PBTX and norepinephrine contracted rat vas deferens. Prazosin 10(-6) mol/L blocked the response to PBTX (3 micrograms/ml) (88.3% to 27.3% contraction [n = 6; p less than 0.001]) and the response to norepinephrine (EC50 was shifted from 1.67 X 10(-6) mol/L to 1.25 X 10(-4) mol/L in the presence of prazosin 10(-6) mol/L [n = 6; p less than 0.001]). Phentolamine 10(-6) mol/L also blocked both PBTX and norepinephrine. Tetrodotoxin 10(-7) mol/L, a sodium channel blocker, completely blocked the response to PBTX but not to norepinephrine. The response to PBTX was significantly reduced from 1.53 gm of tension in controls to 0.29 gm of tension (n = 6; p = 0.002) in tissues obtained from rats pretreated with reserpine (2 mg/kg per day for 2 days, injected intraperitoneally). Verapamil 10(-5) mol/L blocked the PBTX response, and PBTX caused no contraction in calcium-free media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of serotonin in the rat vas deferens: its influence on contractile responses

Serotonin has been detected in the rat vas deferens. Increase in the serotonin concentration by exposure of the rat vas deferens to L-tryptophan occurs in vitro. p-chlorophenylalanine partly blocks the increase in serotonin concentration induced by tryptophan in vitro but not in vivo. Chronic sympathetic denervation induces an increase in 5-HT concentration. Responses of the vas deferens to transmural stimulation are depressed by pretreatment of rats with p-chlorophenylalanine, and the depression is reversed by incubation in vitro with 5-hydroxytryptophan or serotonin. Serotonin can enhance the response to transmural stimulation at low concentrations but has no effect at higher concentrations. Physostigmine-induced enhancement of the response to stimulation is depressed only by higher concentrations of serotonin. The results raise the question whether endogenous serotonin can act as a modulator of neurotransmission in the rat vas deferens.     

Co-transmission in the rat vas deferens: postjunctional synergism of noradrenaline and adenosine 5'-triphosphate

In the isolated prostatic half of the rat vas deferens, joint application of noradrenaline (NA) and adenosine 5'-triphosphate (ATP) produced a contractile response whose magnitude was greatly larger than the addition of the tension generated by the application of each agent alone. The effect of ATP was mimicked by two non-hydrolyzable ATP analogs, but not by GTP, AMP or adenosine. In sympathectomized rats, ATP potentiated NA effects, increasing both the peak tension and the duration of the vas deferens contractile response. The synergism was concentration related. Prazosin antagonized the NA synergism but not the ATP response. Likewise, desensitization of the P2-purinoceptor blocked the ATP synergism without modifying the NA-induced contraction.     

Release of 3H-noradrenaline from the rat vas deferens under various in vitro conditions

The release of ³H-(-)-noradrenaline (NA) from rat vas deferens in vitro was examined under various experimental conditions. It was found that in normal and reserpinized vas deferens the release of NA evoked by (+)-amphetamine (5 times 10^?6 M) or low external Na⁺ (26 mM) was antagonized by imipramine methiodide and desipramine, inhibitors of the NA uptake, but was not dependent on the presence of Ca²⁺ in the medium and was not antagonized by the potent local anaesthetic agent bethoxycaine. The release evoked by veratridine in reserpinized tissue was antagonized by the uptake inhibitors but was in normal tissue only partially inhibited in presence of Ca²⁺ but almost completely in absence of Ca²⁺. The release by high K⁺ (117 mM) + low Na⁺ (26 mM) in normal tissue was dependent on the presence of Ca²⁺ and was antagonized by the muscarinic agonists carbacholine and metacholine and by high concentrations of desipramine. In the reserpinized vasa the corresponding release was not dependent on Ca²⁺ and was not antagonized by the muscarinic agents but was inhibited by high concentrations of desipramine.