Stimulation-evoked Release Research Articles

Age-related changes in adrenergic neuronal function of rabbit vascular smooth muscle

The adrenergic control of vascular smooth muscle was compared in young and adult rabbits using a variety of in vitro techniques. Norepinephrine (NE) content and accumulation of 3H-NE were not different in blood vessels from the two age groups. In contrast, stimulation-evoked release of endogenous NE was reduced by 40–60% in vessels from the aged animals. Functional studies of smooth muscle contractions were carried out using isolated ring segments of the ear artery. There were no differences in the resting force-response relationship between vessels from young and adult rabbits. Maximum contractile responses to nerve stimulation, NE or KCl were not different in vessels from the two age groups, nor was the NE ED 50. However, blockade of the neuronal uptake system with desmethylimipramine produced a greater shift in the NE concentration-response curve in vessels from the young animals compared to the shift in vessels from adult animals. This observation reflects a decline in neuronal NE uptake with age. Although maximal contractile responses to transmural nerve stimulation at 16 Hz were unchanged, responses to stimulation at lower frequencies were reduced in vessels from adult rabbits, an effect which was also enhanced when an antagonist of neuronal uptake was present. Thus, there is a decline in function of adrenergic nerves in adult animals, reflected in a decrease in stimulation-evoked NE release and a decrease in norepinephrine uptake revealed by functional studies. These two effects tend to balance each other, so that there is a small decrease in contractile response to adrenergic nerve stimulation which is exacerbated when neuronal uptake mechanisms are blocked.

Mechanism of presynaptic actions of adenosine and acetylcholine on noradrenaline release in the guinea-pig heart

Isolated heart of the guinea-pig was stimulated transmurally (1 Hz), or by excess [K +] to evoke release of [ 3H]noradrenaline. The interactions of tetraethylammonium, acetylcholine and adenosine were investigated on [ 3H]noradrenaline overflow at different [Ca 2+] concentrations. One mM tetraethylammonium doubled [ 3H]noradrenaline overflow, and at 30 mM the overflow was facilitated by about 50-fold. The facilitatory effect of 30 mM tetraethylammonium gradually decreased with reduction in [Ca 2+] concentration. In 0.1 mM [Ca 2+] overflow of [ 3H]noradrenaline was completely blocked. However, addition of 30 mM tetraethylammonium to 0.1 mM [Ca 2+]-Krebs solution greatly facilitated the overflow. 35 mM K-induced [ 3H]noradrenaline overflow was facilitated by about 5-fold with 20 mM tetraethylammonium, and the facilitatory effect was blocked by 0.37 μM tetrodotoxin; 75 mM K-induced overflow was facilitated by only 1.5-fold by tetraethylammonium. Electrically induced overflow of [ 3H]noradrenaline was significantly reduced (30%) by 0.13 μM acetylcholine, and the effect increased up to about 75% with higher concentrations of acetylcholine (1.3 μM). 1.3 μM acetylcholine reduced 35 mM K-induced overflow by 30% and had no effect on 75 mM K-induced overflow. 0.37 μM tetrodotoxin had an almost similar effect on K-induced overflow. Ten-times higher concentrations of acetylcholine were needed to obtain over 70% inhibition of overflow produced by low or high K. The inhibitory effect of acetylcholine on [ 3H]noradrenaline overflow by 1 Hz was antagonized by increasing concentrations of tetraethylammonium (3 to 30 mM) in 2.5 mM Ca. The antagonism between acetylcholine and tetraethylammonium persisted even when Ca was lowered to 0.02 mM. However, if the overflow was enhanced by other means (high [Ca 2+] plus phentolamine) to the same extent as with tetraethylammonium and low [Ca 2+], then acetylcholine blocked overflow by 75%. 3.7 μM adenosine reduced [ 3H]noradrenaline overflow by about 54%, and this effect was completely prevented by 30 mM tetraethylammonium, either in 2.5 or 0.02 mM Ca. It is proposed that acetylcholine and adenosine interfere with nerve stimulation-evoked release of [ 3H]noradrenaline, presumably by altering electrical properties (i.e. conduction, resting membrane potential, duration of nerve action potential, etc.) of cardiac sympathetic nerve terminals. This primary action would subsequently lead to a reduction in the availability of [Ca 2+] for the release process.

Inhibition by apomorphine of the potassium-evoked release of [3H]-gamma-aminobutyric acid from the rat substantia nigra in vitro.

1 The spontaneous and potassium-evoked release of tritium from the rat substantia nigra prelabelled with [(3)H]-gamma-aminobutyric acid [(3)H]-GABA were assessed in vitro under conditions of superfusion.2 Kainic acid lesions performed in the right caudate nucleus resulted in a 70% reduction in the ability of the homolateral nigral cells to take up and retain [(3)H]-GABA when compared with the unlesioned side. The potassium-evoked release of [(3)H]-GABA remained proportional to the radioactivity retained in the tissue suggesting that the nigral GABA neurones that survived kainic acid treatment were still functional.3 The spontaneous outflow of [(3)H]-GABA was significantly increased by exposure to different concentrations of exogenous GABA (10 to 1000 muM) when amino-oxyacetic acid was present in the incubation medium.4 Apomorphine in concentrations ranging from 1 to 30 muM inhibited the calcium-dependent release of [(3)H]-GABA induced by 1 min exposure to 30 mM K(+). These concentrations of apomorphine did not affect the spontaneous outflow of radioactivity. In vivo administration of haloperidol 0.2 mg/kg antagonized the in vitro inhibition by apomorphine of the K(+)-evoked release of [(3)H]-GABA.5 The results obtained with apomorphine and haloperidol suggest the presence of presynaptic dopamine-like inhibitory receptors in gabaergic nerve terminals.6 Dopamine in concentrations ranging up to 300 muM did not modify either the spontaneous or the K(+)-evoked release of [(3)H]-GABA from the substantia nigra. These concentrations of dopamine effectively displaced [(3)H]-dopamine recently taken up into the substantia nigra.7 Our results do not support the view that dendritic release of dopamine from the substantia nigra might be involved in the physiological modulation of the spontaneous or the stimulation-evoked release of GABA.

Angiotensin II potentiates responses of the rabbit basilar artery to adrenergic nerve stimulation

Angiotensin II has little contractile effect on the isolated rabbit basilar artery; however, it markedly potentiates contractile responses to adrenergic nerve stimulation. This is not a post-synaptic effect of angiotensin, as responses to exogenous norepinephrine are not altered. Angiotensin increases stimulation-evoked release of norepinephrine, and this effect probably accounts for the increased response to adrenergic nerve stimulation. Since sympathetic stimulation may protect the cerebral circulation from hypertensive damage, increased responsiveness to adrenergic nerve activity produced by angiotensin may have a beneficial effect.

Hypogastric nerve from guinea-pig releases prostaglandin-like substance

1. 1. The isolated hypogastric nerve preparation cut out from guinea-pig hypogastric nerve-vas deferens preparation released spontaneously a substance which contracted the rat stomach fundus strip. 2. 2. The fundus-contracting substance has no histamine-, acetylcholine- or serotonin-like nature. The prostaglandin antagonist PPP inhibited the muscle-contracting effect of this substance. 3. 3. The arachidonic acid increased while indomethacin blocked both spontaneous and electrical stimulation-evoked release of fundus-contracting substance. 4. 4. It is concluded that preterminal sympathetic nerve structures involving ganglion cells form and release a prostaglandin-like substance of the E type.

Inhibition by acetylcholine of the stimulation-evoked release of [ 3H]acetylcholine from the guinea-pig myenteric plexus

The longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum was incubated with [ 3H]choline and then superfused with Tyrode's solution. Exposure to [ 3H]choline resulted in the formation of [ 3H]acetylcholine which was released upon electrical field stimulation. The effects of exogenous acetylcholine, physostigmine and scopolamine on the stimulation-evoked release of [ 3H]acetylcholine were studied. In the absence of a cholinesterase inhibitor exogenous acetylcholine (10 −5 M) only slightly inhibited (by 26%) the evoked release of [ 3H]acetylcholine. If the cholinesterase activity of the preparation was reduced by about 50% in the presence of 10 −7M physostigmine, exogenous acetylcholine caused a concentration-dependent depression of the release evoked at 1 Hz. At a concentration of 10 −5 M acetylcholine the release was reduced by 76%. Scopolamine (10 −9 M) shifted the concentration-response curve for the inhibitory action of acetylcholine in a parallel manner to the right. From the dose ratio a pA 2 value of 9.8 for scopolamine against the release-inhibitory effect of acetylcholine was calculated. Physostigmine also inhibited the stimulation-evoked release of [ 3H]acetylcholine in a concentration-dependent manner, the maximal effect measured being an 85% reduction by 10 −5 M physostigmine. In the absence of a cholinesterase inhibitor scopolamine enhanced the evoked release of [ 3H]acetylcholine. The facilitatory effect was more marked at a stimulation frequency of 3 Hz (2-fold increase) than at 1 Hz (1.4-fold increase). It is concluded that extracellular acetylcholine decreases the stimulation-evoked release of neuronal acetylcholine. This inhibition is specifically mediated by a stimulation of presynaptic muscarinic receptors. The increase by scopolamine of the evoked release of [ 3H]acetylcholine suggests that previously liberated acetylcholine may trigger the negative feedback mechanism of acetylcholine release even if the cholinesterase activity is not inhibited, and that the presynaptic muscarinic receptors of the myenteric plexus have a physiological role in regulating the release of acetylcholine.

Inhibition of neuronal uptake reduces the presynaptic effects of clonidine but not of α-methylnoradrenaline on the stimulation-evoked release of 3H-noradrenaline from rat occipital cortex slices

The iminoimidazolidine clonidine reduced concentration-dependently the release of 3H-noradrenaline evoked by electrical stimulation from the rat cerebral cortex. Exposure to the neuronal uptake inhibitors cocaine (10 μM), desipramine (0.1 to 1 μM) and amphetamine (1 μM) significantly increased the stimulation-evoked overflow of tritium. These uptake inhibitors antagonized the effects of clonidine on stimulation-evoked 3H-noradrenaline release but failed to modify the inhibition induced by the catecholamine α-methylnoradrenaline. Inhibition of monoamine oxidase by preincubation of cerebral cortex slices with 0.5 mM pargyline significantly increased the stimulation-evoked overflow of tritium, but clonidine was as effective as in the controls in inhibiting 3H-noradrenaline overflow. The antagonism by desipramine of the clonidine-induced inhibition of neurotransmission could not be attributed to a blockade of presynaptic α-adrenoceptors because: (1) the facilitating effect of phentolamine on 3H-noradrenaline overflow was not modified in the presence of desipramine; (2) the magnitude of the inhibition of the stimulation-evoked 3H-noradrenaline release elicited by α-methylnoradrenaline was the same in the presence of cocaine or desipramine; (3) exposure to desipramine in the presence of cocaine did not further increase the stimulation-evoked release of 3H-transmitter. Since the catecholamine α-methylnoradrenaline inhibited neurotransmission in the presence of desipramine or cocaine, we can conclude that inhibition of neuronal uptake of noradrenaline antagonized selectively the presynaptic inhibitory effects of imidazolines on α 2-adrenoceptors. The influence of the inhibition of neuronal uptake on the presynaptic effects of imidazolines and catecholamines should be taken into account when the relative order of potencies of various α 2-adrenoceptors agonists is determined.

Angiotensin II facilitates the potassium-evoked release of 3H-noradrenaline from the rabbit hypothalamus

Angiotensin II facilitated in a concentration-dependent manner the potassium-evoked 3H-noradrenaline overflow from the rabbit hypothalamus. This effect which is probably mediated through presynaptic angiotensin facilitatory receptors on noradrenergic nerve terminals was blocked by the specific angiotensin receptor antagonist, saralasin. These results demonstrate that angiotensin II also facilitates the stimulation-evoked release of noradrenaline in the central nervous system.

Neuroleptic binding and their effects on the spontaneous and potassium-evoked release of 3H-dopamine from the striatum and of 3H-noradrenaline from the cerebral cortex

Neither dopamine agonists nor neuroleptics modified the stimulation-evoked release of 3H-dopamine from rat striatal slices. In contrast the stimulation-evoked release of 3H-noradrenaline from slices of rat occipital cortex was enhanced by neuroleptics through a blockade of presynaptic α-adrenoceptors. 3H-neuroleptic binding shows two binding sites in both rat striatum and cortex.

Inhibition of noradrenaline release by adenosine.

1. Release of [3H]noradrenaline evoked by stimulation (5 Hz) of the pre-labelled rat vas deferens was reduced to 50% by adenosine (10(-6) g/ml.). Inhibition of release was dependent on the concentration of adenosine and was inversely related to the frequency of stimulation. Phenoxybenzamine did not interfere with the action of adenosine on release. 2. Exposure of the pre-labelled rat vas deferens to 135 mM-K+ released almost 10 times more [3H]noradrenaline than exposure to 45 mM-K+. Release induced by 45 mM-K+ was almost abolished by adenosine (10(-6) g/ml.) but that induced by 135 mM-K+ was reduced to only 45%. 3. Inhibition of [3H]noradrenaline release was observed in salivary gland, heart and portal vein of the rat, and in the guinea-pig heart and vas deferens. A very high concentration of adenosine (10(-4) g/ml.) reduced the release (about 50%) in the rabbit heart, but the cat heart was totally insensitive to the inhibitory action of adenosine. 4. Aminophylline (2 x 10(-4) g/ml.) antagonized the inhibitory action of adenosine (10(-6) g/ml.) on the release of [3H]noradrenaline in the phenoxybenzamine-treated vas deferens. 5. Tetraethylammonium (8 x 10(-4) g/ml.) enhanced stimulation-evoked release in the rat salivary gland by almost tenfold. In the presence of tetraethylammonium, even higher concentrations (2 x 10(-5) g/ml.) of adenosine failed to interfere with release. 6. Elevation of external K+ (19 mM) blocked stimulation-evoked release in the rat vas deferens by about 55%. Combination of high K+ and adenosine (10(-6) g/ml.), which blocked release by about 40%, caused still greater inhibition (80%) of the release. 7. The possible mechanism of action of the inhibitory effect of adenosine on the stimulation-evoked release of noradrenaline is discussed in relation to the calcium hypothesis.

Forces produced by medial gastrocnemius and soleus muscles during locomotion in freely moving cats.

5. During the step cycle the peaks of both MG and SOL force profiles occur before the end of the yield (E2) phase, as both muscles undergo active lengthening. In contrast, peak MG and SOL forces during vertical jumping coincide with the end of active lengthening and the onset of ankle extensor shortening. 6. The results are consistent with the hypothesis that the inherent stiffness of active muscle during the step cycle is an important factor in the control of force output from hindlimb extensor muscles in locomotion. The division of labor between MG and SOL and the absolute force levels required from the MG during the full range of hindlimb movements in posture, locomotion, and jumping appear to be precisely matched to the very different characteristics of the motor-unit populations composing these synergistic muscles.

Isometric force production by motor units of extensor digitorum communis muscle in man

Isometric force production by motor units of extensor digitorum communis muscle in man

Release of 3H-acetylcholine from isolated guinea pig ileum. A radiochemical method for studying the release on the cholinergic neurotransmitter in the intestine.

Innervated strips of longitudinal muscle of guinea pig ileum were incubated with 3H-choline in the presence of physostigmine, after which the efflux of tritiated metabolites was studied. Under resting conditions 3H-acetylcholine, 3H-choline and other tritiated metabolites were released into the incubation medium. Analysis of the kinetics of the release of the metabolites indicated that the release occurred from at least 2 different compartments. Under the unstimulated conditions used, between 72 and 78% of the radio-activity remained in a third, non-releasable compartment. The release of 3H-acetylcholine showed spontaneous fluctuations which were positively correlated to spontaneous variations of the isometric tension of the preparation. Electrical field stimulation at low frequency (0.5 Hz) consistently increased the release of 3H-acetylcholine. The release of 3H-choline was increased only slightly or not at all by the electrical stimulation. For studying stimulation-evoked release of 3H-acetylcholine, it is suggested that the measurement of the total quaternary amine release is of similar value to measurement of 3H-acetylcholine itself. This approach considerably simplified the method. During 5 consecutive stimulation periods of 150 pulses each, the increase in the release of total quaternary amines diminished with time. By taking into account the reduction of the stores of radioactivity in the tissue during the experiment, correction for the negative trend could be made. When the preparation was stimulated with between 30 and 3000 pulses a close correlation was found between the increase in the release of total quaternary amines and the number of pulses delivered. The volley output of the quaternary amines was not constant, however, but diminished by increasing stimulation frequency.

3H-noradrenaline outflow induced from isolated adventitia and intima-media of rabbit aorta by electrical field stimulation

Electrical field stimulation of the isolated adventitial and intima-medial layers of rabbit aorta preloaded with 3H-noradrenaline elicited a 3H outflow. The initial 3H outflow from adventitia was independent of external Ca 2+ concentration. Each of the subsequent 3H outflows was mainly Ca 2+ sensitive. All stimulation-induced 3H outflows from media were Ca 2+ insensitive. It is concluded that the isolated adventitia is a suitable preparation to study electrical field stimulation-evoked release of 3H-noradrenaline. Furthermore, the stimulation-induced 3H outflow from aorta is of neuronal and extraneuronal origin.

Inhibition of adrenergic neurotransmission by prostaglandin E 1 (PGE 1) in the rabbit ear artery

Isolated central ear arteries of rabbits were perfused with Krebs solution at a constant flow rate of 2–7 ml/min. Pressure changes to periarterial nerve stimulation (1, 3, 10 and 30 Hz; 5–32 pulses) and to noradrenaline were measured in the presence and absence of prostaglandin E 1 (PGE 1). This substance in concentrations of 7 × 10 −9– 4.5 × 10 −7 produced a dose-dependent inhibition of constrictor responses to nerve stimulation. The degree of inhibitory action of PGE 1 became progressively less as the frequency or length of stimulation was increased. Prostaglandin E 1 (1.1 × 10 −7 M) failed to affect the constrictor responses to noradrenaline and it markedly reduced the stimulation-evoked release of radioactivity from arteries incubated with [ 3]noradrenaline. The results indicate that PGE 1 inhibits the vasoconstrictor responses of rabbit ear artery to nerve stimulation by reducing the release of the adrenergic transmitter.

Effect of calcium on the relationship between frequency of stimulation and release of noradrenaline from the perfused spleen of the cat.

The sympathetic nerves of the cat spleen were stimulated electrically (20 v, 1 msec, 7 sec) with increasing frequencies (1-30 Hz) during perfusion of the spleen with normal or low-calcium Krebs solution. Noradrenaline (NA) output per stimulus was measured. In spleens perfused with normal Krebs solution, the NA output per stimulus at 5Hz was barely detectable, while the output at 30 Hz was about 5-fold greater than at 5 Hz. Reduction of the calcium concentration in the Krebs solution to 0.2-0.5 mM, while reducing outputs at both frequencies, resulted in an output at 30 Hz that was nearly 8 to 10 times that at 5 Hz. In spleens pretreated with phenoxybenzamine (PBZ) the NA output was increased at all frequencies, and the maximal output occurred at 5 Hz, whereas the output at 30 Hz was only one-third of the maximal output. Reduction of the calcium concentration of the perfusion solution to 0.5 mM, while reducing outputs from PBZ-treated spleens, increased the output at 30 Hz by about 5-fold over the output at 5 Hz. The muscarinic inhibition of stimulation-evoked release of NA by acetylcholine was much more pronounced at low than at high frequency of stimulation. The increase in the release of NA per stimulus with increase in the frequency of sympathetic nerve stimulation is explained on the assumption that the specific entry of calcium ions required for transmitter release is also frequency-dependent.

Nigro-striata pathway: stimulation-evoked release of [ 3H dopamine from caudate nucleus

Nigro-striata pathway: stimulation-evoked release of [ 3H dopamine from caudate nucleus