Part III
Evidence for Cholinergic Inhibitory Innervation of the Cardioarterial Valves
Summary
1.
2.
3.
4.
5.
6.
7.
8.
In the isopod Bathynomus doederleini, thirteen arteries arise from the heart, which are an anterior median artery, a pair of anterior lateral arteries and five pairs of lateral arteries Anterior lateral arteries and lateral arteries receive inhibitory innervation from the central nervous system.
Pharmacological and ionic mechanisms of inhibitory neuromuscular transmission in the valve muscle cells were studied by means of intracellular recording from valve muscle cells and recording of arterial pressure.
Application of acetylcholine (ACh) to the valve increased arterial pressure.
Increase of arterial pressure, which was caused by stimulation of valve dilator nerves, was antagonized by atropine and methylxylocholine.
ACh effectively hyperpolarized muscle cells of all the valves of the anterior lateral artery and lateral arteries which receive inhibitory innervation, but did not affect muscle cells of the valve of the anterior median artery. The muscarinic agonists, muscarine, carbamylcholine and arecoline, mimicked hyperpolarizing responses of the valve.
Atropine, methylxylocholine, tetraethylammonium, 4—aminopyridine and quinine antagonized both inhibitory junctional potentials (IJPs) and the ACh—
induced hyperpolarizing potentials. d—Tubocurarine antagonized the ACh—
induced potentials, but did not IJPs
Escrine increased the amplitude and duration of IJPs.
IJPs and ACh—induced potentials did not invert in Cl--free saline. In low K+
salines, their amplitude was increased, and decreased in high K+ salines. IJPs may be K+—mediated cholinergic responses.
ACh potentials were suppressed by Ca2+—free saline and by Ca2+ channel
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-9.
blockers. Ca2+ may activate K+ efflux in ACh—induced potentials in the valve muscle cells.
Depletion of external Na+ ions resulted in an increase of resting membrane potential. High Na+ conductivity may contribute to the relatively low value of the resting membrane potential of valve muscle cells.
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-Introduction
It is well known that hacmolymph distribution to destinations of arteries is accomplished by neural and humoral control of the vasomotor system in mammals and molluscs. However, in crustaceans, the haemolymph distribution is controlled by the cardioarterial valves located at the junction between the heart and individual arteries, innervated from the central nervous system (CNS) via valve nerves (reviewed by McMahon and Burnett 1990) . In the isopod Bathynomus doederleini, three anterior arteries (AAs) and five pairs of lateral arteries (LA1-5) arise from the heart. AAs consist of an anterior median artery (AMA) and a pair of anterior lateral arteries (ALAs). All the cardioarterial
valves are innervated by their own valve nerves from central ganglia . Kihara
et al. (1985) demonstrated that, in Bathynomus, the cardioarterial valves of the AMA and the ALAs receive excitatory valve nerves, and the valves of the ALA and the LAs receive inhibitory valve nerves. The excitatory valve nerve brings about contraction of a pair of valve flaps, resulting in a decrease of arterial haemolymph flow (Kihara and Kuwasawa 1984; Kihara et al. 1985). On the other hand, the inhibitory valve nerve causes dilation of the valve, resulting in an increased flow (Kihara and Kuwasawa 1984; Kihara et al. 1985; Fujiwara—
Tsukamoto et al. 1992).
In Bathynomus, acetylcholine (ACh) may be the inhibitory neuromuscular transmitter of the valve dilator nerves (Kihara and Kuwasawa 1985, Fujiwara and Kuwasawa 1987). This study was carried out to obtain convincing evidence for cholinergic inhibitory innervation of the cardioarterial valves of Bathynomus doederleini, and to characterize ionic mechanisms of the valve muscle cells.
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-Materials and Methods
Animals
Specimens of Bathynomus doederleini, 9-15 cm in body length, were collected and kept in the laboratory's aquarium as described in Part I.
Dissection and preparations
For intracellular recording, the basal part of the artery, which contained a pair of flaps of the cardioarterial valve, was dissected out together with the valve nerve.
The specimens were carefully incised along a long axis of the artery in order to expose the valve, and pinned inside out to the bottom of a Sylgard—lined experimental bath (0.5 ml). Preparations, used for recording of the arterial pressure, consisted of the arterial system and dorsal body wall. They were pinned to the bottom of a soft plastic—lined bath (30 ml). Since some cholinergic agonists and antagonists induced excitatory effects on the heart, I used preparations from which heart tissues were completely removed. Arterial saline flow was artificially produced by direct supply of perfusate to the artery, using a cannula whose tip was positioned near the junction between the heart and the artery. Artificial sea water (ASW) was continuously applied to the chamber, through a temperature—controlling water bath unit (Model CTC-100, Aqua, Tokyo, Japan), by gravity—feeding, and then suctioned for removal. ASW, containing drugs, was superfused to preparations in the chamber by means of turning the switch of a three—way valve at flow rates of 0.7-2.1 ml/min for intracellular recording, and of 2.2-3.2 ml/min for pressure recording.
Temperature in the chamber was kept at 17-20 °C.
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-Electrophysiology
Intracellular recording from the valve muscle cells was carried out with glass microclectrodes filled with 3 M KCI (tip resistance, 8-40 MQ). The intracellular microelectrode, connected to an Ag—AgC1 wire, was coupled to a high input impedance preamplifier (Nihon Kohden, MEZ-8201). In the case of C1--free saline, an Ag—AgC1 reference electrode communicated with a 3 M KC1 reservoir and an agar bridge. The agar bridge was made of glass with a 1.4 mm inside diameter, filled with 2% agar dissolved in 3 M KCI. A tip of the agar bridge was positioned at the site where perfusate was removed by suction. Arterial pressure was recorded with a pressure transducer (Gould, P-50). The probe of the transducer was connected to a polyethylene cannula (0.25 mm in inside diameter) whose tip was inserted into the artery. Signals were displayed on a pen—writing chart recorder (Nihon Kohden, PMP-3004) and on a CRO (Tektronix, 5110).
Signals on the CRO display were continuously photographed by a CRO camera (Nihon Kohden, RLG-6101).
ACh was applied iontophoretically by using a glass micropipette filled with 1 M ACh (tip resistance, 15-80 MQ). Positive current pulses (15-150 nA, 40-200 msec in duration) from a pulse generator (Nihon Kohden, SEN-3101) were applied to an ACh micropipette connected to an Ag—AgC1 wire. A negative retaining current of about 5 nA was continuously applied to the micropipettes.
The tip of the ACh pipette was positioned as close to a recording microelectrode as possible. For stimulation of valve nerves, the distal cut—stump of the nerve was introduced into a glass suction electrode connected to an Ag—AgC1 wire. A stimulating electrode was connected to a pulse generator via a photic isolator (Nihon Kohden, SS-201J).
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-Composition of solutions
The ionic composition of the ASW employed in this study was almost the same as reported by Yazawa and Kuwasawa (1984). Ionic compositions of various kinds of salines are shown in Table 1. Low K+ salines were prepared by reducing the concentration of KC1. High K+ salines were prepared by adding KC1 and removing NaCI to preserve osmolarity. For making solutions at various concentrations of Na+, Tris (hydroxymethyl) aminomethane hydrochloride was substituted for NaCl. Cl--free saline was prepared by substituting propionate and sulfate, or acetate and sulfate for Cl-. For Ca2+—free saline, MgC12 was substituted for CaC12. Tris (hydroxymethyl) aminomethane was added to adjust to pH 7.5-7.8.
Chemicals
The following blockers were used: atropine sulfate monohydrate (Wako Pure Chemical), methylxylocholine chloride (also called 13—methyI TM 10, a gift from Smith, Kline & French Laboratories, Philadelphia, Pennsylvania), d—tubocurarine chloride (Wako Pure Chemical), picrotoxin (Wako Pure Chemical), bicuculline methobromide (Sigma), tetraethylammonium chloride (Kanto Kagaku), 4—aminopyridine (Wako Pure Chemical), quinine hydrochloride (Wako Pure
Chemical), verapamil hydrochloride (Sigma), acetylcholine chloride (Wako Pure Chemical), dl—muscarine chloride (Sigma), carbamylcholine chloride (Sigma), arecoline hydrobromide (Sigma), nicotine tartrate (Wako Pure Chemical), y—aminobutyric acid (GABA, Wako Pure Chemical), ouabain (Wako Pure
Chemical) and eserine sulfate (Wako Pure Chemical).
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-Results
Effects of ACh and cholinergic antagonists on arterial pressure
ACh increased arterial pressure conspicuously in a dose—dependent manner (Fig. 1A). Figure 1B and C show effects of cholinergic blockers on the increased arterial pressure induced by stimulation of the valve dilator nerve. A representative muscarinic blocker, atropine (10-3 M), effectively blocked the response (Fig. 1B). Methylxylocholine (10-4 M), which is known as a blocker for K+—mediated ACh response in molluscs (a review of Gerschenfeld 1973; Elliott 1979, 1980), also blocked the response (Fig. 1C).
Effect of ACh and cholinergic agonists on the valve muscle
Intracellular potential was recorded from muscle cells of the cardioarterial valves. Resting membrane potential of the valve of LA5 ranged from —23.0 to
—39.0 mV. Mean resting membrane potential with standard deviation (S. D.) was
—30.0±4.1 mV (N=16). Figure 2 shows effects of ACh on all the cardioarterial valves, i. e. the valves of the AMA, the ALA and LAs 1-5. Bath—applied ACh (10' M) hyperpolarized the valves of the ALA and LAs 1-5 by 20-30 mV.
These indicate that the valves of the ALA and LAs 1-5 have hyperpolarizing ACh receptors. It was noticeable that the valve of the AMA did not respond to even
high concentrations (up to 10-4 M) of ACh (N=3). This may reflect that the valve of the AMA receives only excitatory innervation from the CNS (Kihara et al.
1985).
LA5 is the artery sending its branches to the swimmerets. The valve of LA5 receives innervation from the 5th lateral cardiac nerve (LCN5) which consists of two valve inhibitory axons (Fujiwara—Tsukamoto et al. 1992). Hyperpolarizing
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-potentials were induced by perfusion with ACh at 10-10 to 10-5 M in a dose—
dependent manner (Fig. 3). The threshold concentration of the valve of LA5 for response to application of ACh to the bath was between 10-10 M and 10-9 M.
Two different sizes of discrete inhibitory junctional potentials (IJPs) are evoked in the valve of LA5, by the stimuli to LCN5 in two different stimulus intensities (cf. Kihara et al. 1985). These are small sized single unit IJPs and large sized compound IJPs (see Fig. 6). Compound IJPs, generated by stimulation of the two LCN5 axons, were 16.8+4.3 mV (mean + S.D., N=17) in size. Figure 4A shows trains of compound IJPs caused by stimulation of LCN5 at various frequencies (0.5, 1 and 10 Hz). The resting membrane potential of the valve cell (Fig. 4A) was —35 mV. The membrane potential of the valve reached —66 mV at 10 Hz.
When the specimen was perfused with 5x10-7 M ACh during trains of compound IJPs (Fig. 4B), membrane potential was hyperpolarized from —30 mV to —57 mV, and the amplitude of the IJPs was reduced, depending on the hyperpolarizations.
However, hyperpolarizing IJPs were never inverted into depolarizing potentials.
While recording the membrane potential, constant negative current pulses were applied through another microelectrode which was inserted into a muscle cell less than 200 p.m apart from the recording electrode. 10-6 M ACh reduced pulse amplitude by 12.3 % at the most hyperpolarized level (-59 mV) (Fig. 4C).
I examined effects of some ACh agonists, muscarine, carbamylcholine and arecoline, on the valve of LA5 (Fig. 5). These agonists showed actions similar to ACh. Application of each agonist to the bath at 10-5 M induced 25-30 mV of hyperpolarization of the valves, which is similar extent of hyperpolarization to that induced by 10-' to 10-6 M ACh (Fig. 3). Nicotine (10-4 M) caused less than a
few mV depolarization, and never caused hyperpolarization of the valve.
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Effects of cholinergic antagonists on IJPs and ACh—induced potentials
Atropine is known as a muscarinic antagonist. Methylxylocholine blocks K+—
mediated ACh responses in gastropod neurons (reviewed by Gerschenfeld 1973) and bivalve heart (Elliott 1979, 1980). d—Tubocurarine, which is known as a nicotinic antagonist, blocks C1--mediated ACh responses in gastropod neurons (reviewed by Gerschenfeld 1973), bivalve heart (Elliott 1979, 1980; a review of Hill and Kuwasawa 1990) and gastropod hearts (Kuwasawa and Yazawa 1980;
Kuwasawa et al. 1987). Figure 6 shows effects of the three cholinergic blockers on IJPs and ACh—induced hyperpolarizing potentials (ACh—potentials). Both unitary and compound IJPs were observed, since one or the other could be elicited by means of changing stimulus intensity. Both IJPs were effectively antagonized by atropine (5x10-4 M). Atrophic (5x10-5 M) antagonized ACh—potentials more effectively than IJPs. Application of methylxylocholine strongly blocked both IJPs at 10-5 M and ACh—potentials at 10-6 M. Administration of d—tubocurarine (up to 5x10-4 M) slightly shortened time course of IJPs, but did not affect amplitude of IJPs. However, the same concentration of d—tubocurarine effectively blocked ACh—potentials.
Eserine effects on IJPs
Figure 7 shows the effects of eserine on IJPs. When applied to the LCNS, a single stimulus, in one case (left panel in Fig. 7A), and three repetitive stimuli, in another case (right panel in Fig. 7A), elicited corresponding IJPs (see control in Fig. 7A). Administration of eserine (10-4 M) increased IJP amplitudes , and elongated the time course of IJPs even though 3 mV of hyperpolarization occurred in this preparation. Eserine is known as a reversible anticholinesterase agent (reviewed by Taylor 1990). However, even with more than 1 hr wash, IJPs did
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-not recover from potentiation (data -not shown). Figure 7B shows the increase of IJP amplitude and of half time of IJP recovery phase. All data were obtained from experiments on preparations which had been perfused with 10-4 M eserine.
Mean % increase with standard error (S. E.) of IJP amplitude and half time of recovery phase were 20.7+8.4 % and 93.7+26.2%, respectively (6 cells in 4 animals).
Ionic mechanisms of IJP and ACh responses
I examined the action of tetraethylammonium (TEA), 4—aminopyridine (4—AP) and quinine, on the valve of LAS. TEA is a general K+ channel blocker. 4—AP is known as a blocker for voltage—dependent K+ channels. Quinine is classified as a blocker for intracellular Ca2+—dependent K+ channels. All the three K+ channel blockers preferentially blocked both IJPs and ACh—potentials (Fig. 8). The results suggest that IJPs of the cardioarterial valve cells may include a K+
component.
I examined the effects of various salines with altered concentrations of Crand K+ ions on IJPs and ACh—potentials. Figure 9 shows an example of results, using a Cl--free solution in which propionate and sulfate were substituted for Cr.
Right after Cr—free saline was applied, the membrane potential of valve muscle cell was hyperpolarized by more than 20 mV. IJPs completely disappeared.
However, both IJPs and ACh—potentials were never inverted into depolarizing potentials. Almost the same results were obtained with a low CL and a CL—free salines, and with the solutions in which acetate and sulfate were substituted for Cl-(data not shown).
Figure 10 shows the effect of lowering external K+ concentrations ([K+]) on IJPs and ACh—potentials. As [K+] was reduced from ASW (11 mM) to lower
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[K+] solutions (down to 1 mM), the amplitudes of both IJPs and ACh—potentials increased. Resting membrane potential increased by only 1 to 2 mV. As is the case with low [K+] solutions, successive changes to higher [K+] solutions (up to 55 mM) did not significantly affect resting membrane potential (Fig. 11). Resting membrane potential acutely depolarized when [K+] was changed to 110 mM. As [K+] was increased, amplitudes of IJPs and ACh—potentials decreased correspondingly. In order to hold the membrane potential at the value in ASW, another microelectrode was inserted for current injection. The holding potential was —33 mV in a column of "holding" of Figure 11A, ACh—potential. At 55 mM K+, ACh—potentials wcre inverted to depolarizing potentials, and at 110 mM K+
their amplitudes became larger. On the other hand, IJPs disappeared in 110 mM K+ probably because of conduction blockage of the valve nerve (see Fig. 11A, the 3rd panel of IJP), even though the membrane potential was held at the value in ASW (data not shown). The inverted ACh—potentials in 110 mM K+ were blocked by atropine (2x10-4 M) and TEA (10-4 M) (Fig. 11B) as were the hyperpolarizing ACh—potentials in ASW (see Figs. 6 and 8).
Contribution of Ca 2+ on ACh —potentials
Ca2+—free saline effectively inhibited ACh—potentials (Fig. 12). The blocking effect of Ca2+—free saline progressed very slowly, and required more than 20 min to reach the maximum inhibition of ACh—potentials. Ca2+ channel blockers, verapamil (5x10-5 M) and cadmium (10-4 M), exerted similar action on the ACh
potentials. These results suggest that Ca2+ may relate to the ACh response of the valve muscle cells.
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-Effects of K+ and Na+ on the resting membrane potential
It was characteristic that the resting membrane potential of valve muscle cells was relatively low, around —30 mV. Figure 13A shows membrane potentials of the valve muscle cells of LAS in various [K+] solutions. Successive lowering of [K+1 from the concentration in ASW (11 mM) caused slight hyperpolarization.
On the other hand, perfusion with 22 mM K+ solution slightly depolarized membrane potential. At 55 mM K+, a transient depolarization occurred at the beginning of perfusion, but the membrane potential stabilized at almost the same value as that in ASW. A solution containing 110 mM K+ suddenly caused more than 10 mV depolarization of membrane potential.
Figure 13B shows effects of various concentrations of [Nat] on resting membrane potential. [Na+] in ASW is 526 mM. Four kinds of low [Nat]
solutions (0, 53, 105, 263 mM) were prepared by substitution of Tris for Nat.
Successive changes of [Nat] from ASW to lower solutions gradually
hyperpolarized the membrane potential. The difference between mean resting
membrane potential in ASW (N=16) and in a Nat—free solution (N=5) was
14.2 mV. The results suggest that Na+ ions contribute to lowering membrane potential of valve muscle cells.
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-Discussion
Cholinergic inhibitory innervation
GABA is well known as an inhibitory transmitter in invertebrates and vertebrates. GABA has been proved to be a transmitter of the extrinsic cardioinhibitory nerve in some crustacean species (reviewed by Yazawa and Kuwasawa 1992). Kuramoto et al. (1992) recently suggested GABAergic inhibitory innervation of the cardioarterial valve of the lobster, Homarus americanus. In this study, even a high concentration of GABA (10-4 M) did not show definite hyperpolarization of membrane potential of the valve muscle cells (data not shown). The GABAergic blockers, picrotoxin (10-4 M) and bicuculline (2x10-4 M), reduced the amplitude of IJPs slightly, by less than a few mV (data not shown). It has been reported in crustacean (Marder and Paupardin—Tritsch 1980), insect (Benson 1988), mollusc (Yarowsky and Carpenter 1978) and annelid (Schmidt and Calabrese 1992) that such GABAergic blockers could antagonize ACh responses. Thus, GABA may not be an inhibitory transmitter of the inhibitory valve nerve of Bathynonzus doederleini.
ACh is an excitatory neuromuscular transmitter in crayfish skeletal muscles (Futamachi 1972) and in the stomatogastric system of the lobster (Marder 1974, 1976). In the cardiovascular system of crustaceans, ACh has been proposed as the transmitter of the extrinsic cardioacceleratory nerve of Bathynonzus doederleini (Tanaka et al. 1992), and as a neurotransmitter of small cells of the cardiac ganglion of the hermit crabs (reviewed by Yazawa and Kuwasawa 1992). In stomach and opener muscles of the crayfish, Zufall et al. (1988) reported the existence of ACh—activated Cl- channels by means of a patch clamp method.
Kihara and Kuwasawa (1985) first suggested that ACh may be the inhibitory
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-transmitter released by the innervation of the cardioarterial valves of Bathynonzus doederleinc. This study presented convincing evidence for cholinergic inhibitory
innervation of the cardioarterial valves. The membrane potential of valve muscle cells was hyperpolarized by ACh in a dose—dependent manner. Effects of muscarinic agonists, cholinergic blockers and eserine revealed the cholinergic nature of inhibitory valve nerves. Results were mainly obtained from the valve of
LAS. However, it is quite likely that the conclusions may be generalized over the valves of all other arteries except for AMA, i. e. valves of the ALA and the LAs 1-4, since all the valves were hyperpolarized (Fig. 2) and dilated (Fujiwara and Kuwasawa 1987) by ACh. This study performed on the cardioarterial valves of Bathynomus may be another example showing that ACh acts as an inhibitory neuromuscular transmitter in the crustaceans.
Ionic mechanisms of IJPs and ACh—induced potentials
Atropine, which is a muscarinic antagonist, effectively blocked both IJPs and ACh—potentials. In the bivalve myocardium, methylxylocholine blocks a K+—
mediated ACh response (Elliott 1979, 1980), and d—tubocurarine blocks a Cl--mediated ACh response of the gastropod myocardium (Kuwasawa and Yazawa 1980; Kuwasawa et al. 1987; reviewed by Hill and Kuwasawa 1990).
In this study, IJPs were also blocked by methylxylocholine, but not by d—tubocurarinc. On the other hand, ACh—potentials were blocked by both methylxylocholine and d—tubocurarine. It may be possible that ACh—sensitive Crchannels are distributed in the extrajunctional region as well as ACh—sensitive K+ channels. Amplitude of both IJPs and ACh—potentials increased and decreased in, respectively, low [K+] and high [K+] saline. Hyperpolarizing ACh—
potentials were inverted into depolarization upon treatment with high [K+]. The
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-inverted ACh potentials were antagonized by atropine and TEA, as were the hyperpolarizing ACh response. If IJPs and ACh—potentials were exclusively C1--mediated, depolarizing responses would be observed in C1--free saline, according to the shift of the Cl- equilibrium potential to a more depolarized level.
However, neither IJPs nor ACh—potentials inverted with C1--free saline, even though membrane potential was intensely hyperpolarized. From these results, it was concluded that IJPs and ACh—potentials are mainly mediated by K+ ions.
Application of a Ca2+—free saline was accompanied by depolarization of membrane potential, and effectively suppressed ACh—potentials. Cadmium and verapamil also blocked ACh—potentials. Since verapamil is a blocker for
transmemhrane Ca2+ current (Kohlhardt et al. 1972), Ca2+ influx might be reduced by the treatment. Existence of intracellular Ca2+—dependent K+ channels has been shown in a wide variety of excitable cells (for reviews, see Meech 1978; Schwarz and Passow 1983; Rudy 1988). The blocking effect of quinine, which is known as an intracellular Ca2+—dependent K+ channel blocker, on IJPs and ACh—
potentials may suggest that the hyperpolarizing ACh response of the cardioarterial valve has a close relationship with the intracellular Ca2+ ions.
Characterization of membrane property
Changes of cxtracellular [K+] did not change resting membrane potential, according to Nernst's equation. Decrease of [Nat] apparently increased resting membrane potential. Transient hyperpolarization was induced by replacing K+—
free saline by higher [K+] saline (data not shown). This suggests the existence of
an electrogenic Na+—K+ pump in the cardioarterial valve, although ouabain
(5x10-4 M) depolarized the resting membrane potential only by a few mV (data not shown). Valve muscle cells may have a high Na+ conductivity, which
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-contributes to relatively low resting membrane potential.
Inhibitory valve nerves of the LAs (LCNs) were tonically active in a resting animal, and altered their impulse rates according to physiological demands of target organs in accordance with a strategy of haemolymph distribution (Part I, 1).
As the valves of the LAs receive only inhibitory innervation with a single or two axons, it means that interaction between the resting tension of valve muscles and the impulse frequency of the LCNs determines the volume of arterial haemolymph flow. Fujiwara—Tsukamoto et al. (1992) demonstrated that LCN stimulation at 10 Hz showed an 8.5 times increase in amplitude of the pressure pulses of the LAs. Stimulation of LCN5 at 10 Hz hyperpolarized membrane potential by more than 30 mV (see Fig. 4A). The following points in the nature of the effectors may enable such a wide—range control of arterial haemolymph flow. 1) The resting membrane potential of valve muscle cells is relatively low (about —30 mV). 2) The ionic equilibrium potential of IJPs may be solely mediated by K+ ions which have the most hyperpolarized equilibrium potential among ion species.
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-FigureLegends
Figure 1.
Effects of ACh (A) and cholinergic blockers (B, C) on arterial pressure in LA5.
A. 100 !Al of 10-7-10-5 M ACh were applied through an inlet tube at arrows.
B, C. Nerve stimulation was applied between arrows. LCN5 was stimulated at 2 Hz.
Figure 2.
Effects of ACh on cardioarterial valves. A valve of the anterior median artery (AMA) was perfused with 10-4 M ACh from the point indicated by an arrow.
Also valves of anterior lateral artery (ALA) and the 1st to 5th lateral arteries (LA1-5), were perfused with 10-' M ACh. Numerals at the beginning of traces and at arrows during the perfusion are membrane potential (mV).
Figure 3.
A dose—response curve showing the relationship between ACh and hyperpolarization of cardioarterial valve muscle cells. •, mean values of hyperpolarizing potential + S. E.; A, mean resting membrane potential + S.E.;
N=5 in 4 or 5 animals. S. E. bars were omitted when S.E. were smaller than the size of symbols.
Figure 4.
A. Trains of IJPs caused by LCN5 stimulation at various frequencies.
Underlining show periods of nerve stimulation. B. Effects of ACh on membrane potential of the valve during the occurrence of IJPs. Stimuli were applied at dots.
C. Effects of ACh on membrane conductance. Upper trace, intracellular recording; Lower trace, current monitor. At the underlining, a period of
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-perfusion with ACh (10-6 M). Fast sweep records are shown at the beginning of the trace and during the ACh treatment.
Figure 5.
Effects of muscarinic agonists on the valve of lateral artery. Underlining shows periods of perfusion with agonists.
Figure 6.
Effects of cholinergic blockers on IJPs and ACh—induced potentials (ACh—
potentials). In experiments with atropinc, both unitary and compound IJPs were induced by, respectively low and high intensity stimuli to the dual axon nerve, LCN5. Other data in this figure and all data in following figures were compound IJPs.
Figure 7.
A. A single stimulus (left panel) and three repetitive stimuli (right panel) produced one and three IJPs (control). Eserine (10-4 M) potentiated IJPs.
B. Histogram shows mean % increases in IJP amplitude and half time of recovery phase. Vertical bars, S. E.
Figure 8.
Effects of TEA, 4—AP and quinine on IJPs and ACh—potentials.
Figure 9.
Effects of CV—free saline. Membrane potential increased during perfusion with C1--free saline.
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-Figure 10.
Effects of low [K+] salines. IJPs and ACh—potentials at various [K+] are shown in A. Data in A are plotted in B. Amplitude of IJPs (•) and ACh—
potential (.), and resting membrane potential (A) are shown in mean value of 5 or
6 trials. All S. E. bars were omitted in the graph since they were smaller than the symbols.
Figure 11.
A. Effects of high [K+] salines on IJPs and ACh—potentials. The resting membrane potential in "holding" were held at —33 mV by means of negative DC current injection. B. Inverted ACh—potentials in 110 mM K+ were blocked by atropine and TEA. Membrane potentials were held approximately at —33 mV.
Figure 12.
Effects of Ca2+—free saline, cadmium and verapamil on ACh—potentials. Data
were obtained at 33 min (Ca2+—free), 6 min (Cd2+) and 23 min (verapamil) after
the beginning of perfusions.
Figure 13.
Resting membrane potentials in various [K+] (A) and [Nat] (B). Plots and bars show mean values + S. E. (N=5-16). Arrows show [K+] and [Na+] of normal ASW. [K+] and [Na+] are shown with the logarithmic scale.
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