[Analysis of the contractions evoked by sympathetic nerve stimulation, and thermal effect on the guinea-pig vas deferens--study as a model for the thermotherapy of benign prostatic hyperplasia].

BACKGROUND: The present study was designed to investigate the mechanism of thermotherapy on benign prostatic hyperplasia (BPH), using the guinea-pig vas deferens as a model for BPH. The components of contractions elicited by electrical field stimulation and nicotine were analyzed, and the thermal effect on the vas deferens was examined. METHODS: The vas deferens was dissected, suspended vertically through two silver ring electrodes, and attached to an isometric transducer. The electrical stimulation of 10 constant current pulses (10 mA) with 0.3 msec in duration of 5, 10, and 40 Hz was achieved under air-gap condition. Drugs were added directly to a 5 ml Magnus tube containing Tyrode solution (36 degrees C) gassed with a 95% O2-5% CO2 mixture. The components of contractions evoked by electrical stimulation and nicotine were investigated by tetrodotoxin (TTX), and blocking agents of alpha 1-adrenoceptors and/or purinoceptors. Thermal effect on electrically evoked contractions was examined at incubation temperature of 25 degrees C (control), 43 degrees C, 45 degrees C, 46 degrees C and 47 degrees C for 1 hour. RESULTS: Nicotine (200 microM) elicited biphasic contractions, which were triggered by corelease of noradrenaline (NA) and ATP (N-ATP) from sympathetic nerve terminals by activation of prejunctional nicotine receptors. NA and N-ATP caused the corresponding contractions, alpha 1 and N-ATP components, respectively. Combined application of prazosin (1 microM) and suramin (50 microM) abolished these contractions. Activation of post-junctional alpha 1-adrenoceptors by NA caused release of ATP from muscle cells to produce the contraction (alpha 1-ATP component), which was sensitive to both suramin and prazosin. N-ATP and alpha 1 components attributed to fast and slow part of the contraction, respectively. Electrical field stimulation caused biphasic contractions which consisted of both neurogenic (TTX-sensitive) and non-neurogenic (TTX-insensitive) components. An increase in stimulation frequency (5 to 40 Hz) increased the neurogenic components, which contained alpha 1 and N-ATP components, as well as the case of nicotine. The non-neurogenic components consisted of alpha 1-ATP, muscle-derived ATP (m-ATP) and unknown substance 'X' components. Nifedipine (10 microM). L-type Ca2+ channel blocker, markedly reduced the contractions induced by bath applied phenylephrine (alpha 1-agonist, 100 microM) but only partially blocked the contractions produced by bath applied ATP (500 microM). The contractile force in amplitude and neurogenic components induced by electrical field stimulation did not change at 43 degrees C, but both declined significantly above 45 degrees C. The neurogenic components at 45 degrees C and 46 degrees C were suppressed to 22 +/- 6% and 14 +/- 3% (mean +/- SD) of control, respectively. All the contractile responses were abolished at 47 degrees C. CONCLUSION: The contractions of the guinea-pig vas deferens evoked by electrical field stimulation consisted of alpha 1, N-ATP, alpha 1-ATP, m-ATP and X components. Sympathetic nerve fibers in the muscles were completely inactivated by thermal exposure at 47 degrees C for 1 hour. The results suggest that the minimal temperature for thermotherapy of BPH should be 47 degrees C.

Analysis of hyposmolarity-induced taurine efflux pathways in the bullfrog sympathetic ganglia.

Hyposmolarity-induced taurine release was dependent on the decrease in medium osmolarity (5-50%) in the satellite glial cells of the bullfrog sympathetic ganglia. Release of GABA induced by hyposmolarity was much less than that of taurine. Omission of external Cl- replaced with gluconate totally suppressed taurine release, but only slightly suppressed GABA release. Bumetanide and furosemide, blockers of the Na+/K+/2Cl- cotransport system, inhibited taurine release by about 40%. Removal of external Na+ by replacement with choline, or omission of K+, suppressed taurine release by 40%. Antagonists of the Cl-/HCO3 exchange system, SITS, DIDS and niflumic acid, significantly reduced taurine release. The carbonic anhydrase inhibitor, acetazolamide, reduced the taurine release by 34%. Omission of external HCO3 by replacement with HEPES caused a 40% increase in the hyposmolarity-induced taurine release. Hyposmolarity-induced GABA release was not affected by bumetanide or SITS. Chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and N-phenylanthranilic acid (DPC), practically abolished taurine release. Blockers of K+ channels, clofilium and quinidine, had no effect on the taurine release. The hyposmolarity-induced taurine release was considerably enhanced by a simultaneous increase in external K+. GABA was not mediated by the same transport pathway as that of taurine. These results indicate that Cl- channels may be responsible for the hyposmolarity-induced taurine release, and that Na+/K+/2Cl- cotransporter and Cl-/HCO3 exchanger may contribute to maintain the intracellular Cl- levels higher than those predicted for a passive thermodynamic distribution in the hyposmolarity-induced taurine release.

Presynaptic inhibition by noradrenaline of the EPSC evoked in neonatal rat sympathetic preganglionic neurons.

Visually identified and electrophysiologically characterized sympathetic preganglionic neurons (SPNs) were recorded using the whole-cell voltage clamp technique in slices of neonatal rat spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the nucleus intercalatus in the presence of strychnine (5 microM) and bicuculline (10 microM). These EPSCs were abolished by the antagonist of AMPA-type glutamate receptors, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 10 microM). Bath applied noradrenaline (NA; 0.5-50 microM) dose-dependently and reversibly decreased by up to around 60% the amplitude of the EPSC, without affecting the holding current. The EPSC depression by NA was not accompanied by a change in EPSC reversal potential (around +5 mV), nor were inward currents generated by pressure application of glutamate affected by NA application. A comparable degree of EPSC depression was also seen with the alpha2-adrenoceptor agonist clonidine (5 microM), and the alpha2A-agonist oxymetazoline (5 microM), while the alpha1-agonist phenylephrine (100 microM) caused only a 22% depression. The EPSC depression caused by NA (10 microM) was completely antagonized by either the alpha-antagonist phentolamine (10 microM) or the alpha2-antagonist idazoxan (2 microM). Conversely, the beta-adrenoceptor antagonist popranolol (5 microM), and the alpha1-, alpha2B- and alpha2C-antagonist prazosin (2 microM) were without effect. These results indicate that activation of presynaptic alpha2A-adrenoceptors on inputs to SPNs decreases glutamate release.

Voltage-dependent potassium currents of sympathetic preganglionic neurons in neonatal rat spinal cord thin slices.

Voltage-dependent potassium currents were analyzed in the visually identified sympathetic preganglionic neurons (SPNs) of neonatal rat spinal cord thin slices by the whole-cell patch-clamp technique. Some of the SPNs were identified by the presence of retrogradely transported fluorescent dye, DiI, injected into the superior cervical ganglion several days prior to experimentation. In a tetrodotoxin (TTX)-containing solution, a step depolarization from the holding potential of -72 mV generated a slow outward current that was suppressed by tetraethylammonium (TEA) and by Ca(2+)-free/2.5 ImM Co2+ solution. Ca(2+)-dependent current consisted of a transient and a sustained components. In a Ca(2+)-free (substituted with Mg2+) solution with TTX and TEA, a step depolarization from a hyperpolarized potential evoked a transient outward current that was blocked by 4-aminopyridine (4-AP). A step hyperpolarization evoked a voltage-dependent inward current, the conductance of which was dependent not only on the membrane potential, but also on the extracellular K+ concentration. Tail current analyses revealed that all of these currents were carried by K+ ions. These results indicate that SPN possesses at least five types of voltage-dependent K+ current, including the delayed rectifier current (IK), Ca(2+)-dependent transient current (IC), Ca(2+)-dependent sustained current (IAHP), A-current (IA) and inward rectifying current (Iu), which may be targets of putative transmitters released from various descending and segmental inputs impinging upon the SPN.

Earlier activation of the distal than the proximal site of the coronary sinus may represent retrograde conduction through AV node: significance of recording of far distal coronary sinus.

During retrograde conduction through an accessory pathway (AP) or the atrioventricular (AV) node, earlier activation of the distal recording site than a more proximal site of the coronary sinus (CS) generally indicates retrograde conduction via a distally located AP. Thus, after successful ablation of a left-sided AP, if the distal CS recording site is activated earlier than a more proximal site retrogradely, it is considered to suggest-in the absence of His-bundle recording or more frequently in the setting of poor recording of the low septal right atrial electrogram-a conduction via a second AP (located more distally), and not conduction via the AV node. Yet, we hypothesized that retrograde conduction through the AV node may activate the far distal site of the CS (CSD) earlier than a more proximal site, as the anterior atrial wavefront, coming retrogradely from the AV node and traveling along the anterior mitral annulus, could reach the CSD earlier than a more proximal site. To test this we studied 18 patients with intact retrograde conduction via the AV node, but without evidence of an AP. The CSD was recorded by means of a quadripolar catheter (interelectrode distance of 2-5 mm); retrograde activation sequence at the distal (CSD1-2) versus proximal (CSD3-4) bipolar recording site was determined during ventricular stimulation. In 12 of 18 patients the CSD1-2 recording site was activated 5-10 ms earlier than the CSD3-4 recording site, in 3 of 18 patients the CSD1-2 site was activated 5 ms later than the CSD3-4 site; in the remaining 3 patients both recording sites were depolarized simultaneously. The results indicate that the CSD was often depolarized earlier than a more proximal site by impulses that conducted to the atria retrogradely via the AV node while the quadripolar recording catheter was placed at the CSD. This observation, although not well documented previously, suggests that the sequence of retrograde atrial activation in the CS should be studied carefully in consideration of the actual location of the mapping catheter in order to correctly diagnose the presence or absence of conduction via an AP.

Characteristics of Na+ current in Schwann cells cultured from frog sciatic nerve.

Characteristics of voltage-dependent currents in cultured frog Schwann cells were investigated by the whole-cell clamp technique. An inward current was detectable at a membrane potential level more positive than -50 mV and reached a maximum value at about -10 mV, while no rectifying channel was present. The inward current was carried by Na+ ions, because the extrapolated reversal potential of the current agreed with the calculated ENa, and the current was sensitive to tetrodotoxin. The membrane potential for half-maximal inactivation was -82 mV. The inactivation curve indicated that more than 90% of the Na+ channels were inactivated at the resting membrane potential, suggesting that the cultured frog Schwann cells could not generate an action potential under physiological conditions. The time constant for the inactivation at a maximum current was 5.3 ms (-10 mV, 13 degrees C). The electrophysiological characteristics of the Na+ current in the cultured frog Schwann cells were compared with those in other tissues. This Na+ current was quantitatively different from that observed in the amphibian node of Ranvier but was similar to that in the mammalian Schwann or glial cells, especially in the more hyperpolarized half-maximal inactivation potential and in the slower inactivation time course.

Phenytoin partially antagonized L-type Ca2+ current in glucagon-secreting tumor cells (ITC-1).

Transmembrane Ca2+ currents were investigated by means of a whole-cell clamp technique in a hamster glucagon-secreting tumor cell line (ITC-1). Two types of Ca2+ current were identified in ITC-1 cells. The low-threshold and transient (T-type) current became detectable above the potential level around -60 mV and decayed rapidly with an inactivation time constant of 95 ms (at -40 mV and 23 degrees C), while the high-threshold and long-lasting (L-type) one was activated by depolarization more positive to -30 mV with non-inactivating kinetics. The voltage dependence and kinetics of these currents were identical to those reported in guinea-pig pancreatic alpha 2 cells. Both currents were augmented by equimolar substitution of Ca2+ with Ba2+ and completely abolished by adding 1 microM La3+. Phenytoin, a well known anti-epileptic drug and a postulated T-type specific Ca2+ current antagonist, surprisingly blocked the L-type current without affecting the T-type current in ITC-1 cells. While phenytoin antagonized the L-type Ba2+ current selectively, 60% of the current remained even in supramaximal concentration range over 500 microM. The residual component of the L-type current was completely abolished by adding nifedipine.

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia.

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased both Km and Vmax for GABA uptake, with an increase in the Vmax/Km ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 microM GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 microM ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 microM veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na+, K(+)-ATPase acting as an ion pump.

Glial uptake system of GABA distinct from that of taurine in the bullfrog sympathetic ganglia.

The kinetics and specificity of GABA and taurine uptake were studied in the bullfrog sympathetic ganglia. GABA uptake system consisted of simple saturable component and taurine uptake system consisted of two saturable components exclusive of non-saturable influx. Taurine unaffected GABA uptake while GABA inhibited taurine uptake competitively with the Ki/Km ratio of 38. GABA (5.14 microM) uptake was inhibited by delta- aminovaleric acid and slightly by 2,4-diaminobutyric acid (5 mM, each) among ten structural analogs. Taurine uptake under high-affinity conditions was most strongly suppressed by hypotaurine and beta-alanine competitively with the Ki/Km ratio of 1.0 and 1.9, respectively. Autoradiography showed that glial cells were heavily labeled by both [3H]GABA and [3H]taurine. These results suggest that GABA is transported by a highly specific carrier system distinct from the taurine carrier and that taurine, hypotaurine, and beta-alanine may share the same high-affinity carrier system in the glial cells of the bullfrog sympathetic ganglia.

Taurine uptake by glial cells in the bullfrog sympathetic ganglia.

Taurine uptake was studied in the bullfrog sympathetic ganglia. High- and low-affinity components were detected after subtraction of nonsaturable influx from total uptake in a concentration range from 34 nM to 8mM. Taurine uptake was strictly sodium dependent and the sodium dependency curve was sigmoidal, with a Hill number of 1.6, indicating that at least two sodium ions are required for the transport of one taurine molecule. External sodium ion affected both K(m) and V(max) for taurine uptake. Taurine uptake was inhibited by ouabain, but not by tetrodotoxin. These results suggest that sodium concentration gradient across plasma membrane may be the main driving force for taurine uptake. Electron and light microscopic autoradiography showed that glial cells were heavily labeled by [(3)H]taurine while ganglion cells were slightly labeled. The present data suggest that glial uptake may contribute to terminating the effect of taurine in the bullfrog sympathetic ganglia.

Membrane properties of preganglionic fibers occurred in bullfrog lumbar sympathetic ganglion.

Membrane properties of preganglionic fibers in the bullfrog lumbar sympathetic ganglion were studied using a conventional intracellular microelectrode technique. Charging or discharging electrotonus of the preganglionic fibers were found to be slow. The slow and fast time constants were around 62 and 15 msec, respectively. An inward-going rectification was activated in the potential range below -80mV. These electrophysiological properties differed from those observed in a principal neuron and suggested a close resemblance between the sympathetic preganglionic fibers and thick myelinated fibers.

Hyperpolarization following activation of K+ channels by excitatory postsynaptic potentials.

We have postulated that an excitatory postsynaptic potential (e.p.s.p.) may open voltage-sensitive K+ ('M') channels, in an appropriate depolarizing range, and that this could alter the e.p.s.p. waveform. Consequently, the fast e.p.s.p. in neurones of sympathetic ganglia, elicited by a nicotinic action of acetylcholine (ACh), could be followed by a hyperpolarization, produced by the opening of M channels during the depolarizing e.p.s.p. and their subsequent slow closure (time constant-150 mg). This introduces the concept that transmitter-induced p.s.ps may trigger voltage-sensitive conductances other than those initiating action potentials, and that in the present case this could produce a true post-e.p.s.p. hyperpolarization. (Some hyperpolarizations other than inhibitory postsynaptic potentials (i.p.s.ps) have been reported to follow e.p.s.ps.) We show here that this is so.

Two muscarinic depolarizing mechanisms in mammalian sympathetic neurons.

A voltage-sensitive outward membrane current ('M') and a consequent change in conductance (delta G) appear with a slow time-constant, in principal neurons of rabbit superior cervical ganglion (SCG), only when membrane potentials (Vm) are depolarized to less than -60 mV. Effects of muscarine on the voltage-current curves indicate that, in this depolarized range of less than -60 mV, suppression of M-current could contribute a muscarinic depolarization accompanied by a decrease in G; but that, at all Vms tested (about -90 to -40 mV), there is an additional larger muscarinic depolarization with no delta G. Thus, the muscarinic depolarizing response and the equivalent slow excitatory postsynaptic potential in the rabbit SCG may consist of two different components: one is due to the suppression of M-current and is substantial only in the depolarized range; the other is probably mediated via an intracellular increase in cyclic GMP and can account for most or all of the response at Vms more negative than -55 mV.

Dopamine as a synaptic transmitter and modulator in sympathetic ganglia: a different mode of synaptic action.

An analysis of the role of adrenergic transmission in mediating the hyperpolarizing, slow inhibitory postsynaptic potential has revealed that dopamine is apparently the specific synaptic transmitter for this response. An additional action of dopamine was discovered, namely the selective facilitation of another synaptic response, the slow excitatory postsynaptic potential. (This potential is a depolarizing response to the muscarinic action of acetylcholine.) This second, modulatory, role of dopamine has characteristics strikingly different from other known modes of synaptic action. After a brief initial action by dopamine, the facilitation of the slow excitatory postsynaptic potential response can persist for hours and is unaffected by a delayed blockade of the postsynaptic receptors for dopamine. This suggests that the modulation consists of a long-lasting metabolic and/or structural change induced in the postsynaptic neuron by dopamine. These conclusions are based on the demonstrated actions of dopamine and other catecholamines, as well as on effects (on dopamine actions and on slow postsynaptic potentials of alpha-adrenergic blockers, of blockade, of dopamine oxidase, of depletion of ganglionic catecholamine by muscarinic excitation, and of a selective re-uptake of dopamine after such depletion.