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.     

Different ion channel mechanisms between low concentrations of capsaicin and high concentrations of capsaicin and nicotine regarding peptide release from pulmonary afferents

Vagal nerve stimulation (1 Hz for 1 min), capsaicin (10^-8 M and 10^-6 M), resiniferatoxin (3 × 10^-10 M) and nicotine (10^-4 M) evoked a non-cholinergic bronchoconstriction in the isolated perfused guinea-pig lung preparation. Simultaneously there was an increase in the perfusate levels of calcitonin gene-related peptide-like immunoreactivity, suggesting release from sensory nerves. Both the bronchoconstriction and peptide release evoked by a low concentration of capsaicin (10^-8 M) and that evoked by nerve stimulation were depressed by tetrodotoxin, suggesting involvement of Na⁺ channel dependent depolarization. Since the effects of capsaicin (10^-8 M) and vagal nerve stimulation were inhibited by ω-conotoxin but not influenced by nifedipine, the Ca²⁺-channel involved is probably of N-type. Furthermore, the capsaicin analogue resiniferatoxin also evoked ω-conotoxin sensitive peptide release and bronchoconstriction. At the higher capsaicin concentration (10^-6 M), the functional response was only slightly inhibited by wconotoxin or tetrodotoxin indicating that capsaicin at this concentration evoked peptide release and functional effects through other mechanisms, probably involving Ca²⁺ fluxes in the non-selective cation channel associated with the proposed capsaicin receptor. The nicotine (10^-4 M) evoked peptide release and bronchoconstriction were only marginally influenced by ω-conotoxin or tetrodotoxin. It is concluded that the ion-channel mechanisms underlying the peptide releasing properties of antidromic nerve stimulation and low concentrations of capsaicin are similar and depend on action potential propagation, whereas capsaicin in high, toxic concentration and nicotine mainly act via receptor operated channels.     

Vascular and Metabolic Effects of Theophylline,Dibuturyl Cyclic AMP and Dibuturyl Cyclic GMP in Canine Subcutaneous Adipose Tissue in situ1

In canine subcutaneous adipose tissue theophylline (2×10^--⁴ M), cAMP and ATP (10^--⁵ M), and DBcAMP (8×10^--⁴ M) increased blood flow by by approximately 100 per cent. These compounds also antagonized sympathetic vasoconstriction. Theophylline and DBcAMP increased glycerol release dose-dependently, while cAMP and ATP were ineffective up to I mM. Theophylline (0.4–0.8 mM) potentiated the lipolytic effect of nerve stimulation, while 2–8 mM apparently caused maximal stimulation Per se. DBcAMP did not affect FFA release following nerve stimulation, while DBcGMP potentiated. The apparent rate of re-esterification, glucose uptake and lactate release was decreased by theophylline. DBcAMP (0.1–0.4 mM) had no effect on these parameters, while DBcGMP at the same concentration decreased re-esterification and lactate release. Stimulated overflow of ³H from tissues prelabelled with L-³H-noradrenaline was reduced to 50 per cent by ATP (0.1–0.4 mM), but was unaffected by DBcAMP and DBcGMP at the same concentration. The results support the view that cAMP mediates the metabolic actions of sympathetic nerve stimulation in canine subcutaneous adipose tissue. The relationship between cAMP and vascular reactions may be more complex.     

Contractile responses of rat duodenum caused by transmural nerve stimulation: interaction between tachykininergic and cholinergic mechanisms

We have studied the importance of tachykinins and acetylcholine for motor stimulation of the rat duodenum in vitro. Contractions induced by transmural nerve stimulation and tachykinin receptor agonists selective for NK1, NK2 and NK3 receptors were used in combination with a neurokinin (NK)2 receptor antagonist, MEN 10,627, and atropine as a muscarinic receptor antagonist. Transmural nerve stimulation in the range 0.5–32 Hz caused frequency-dependent contractions. MEN 10,627 (10^-8, 10^-7 and 10^-6m ) dose-dependently reduced the contractile frequency–response curve (P<0.01–0.001). Addition of atropine (10^-6m ) completely inhibited the response to transmural nerve stimulation (P<0.001). As control, atropine alone reduced this response only by about 65%. Of the tachykinin analogues, [β-Ala⁸]-neurokinin A(4-10) selective for NK2 receptors caused concentration-dependent contractions with high potency (pD₂ 8.01) and high efficacy, while substance P methyl ester acting on NK1 receptors had lower potency (pD₂ 7.94) and low efficacy, and senktide acting on NK3 receptors had a low potency (pD₂ 7.52) but high efficacy. With increasing concentrations of MEN 10,627 the response to [β-Ala⁸]-neurokinin A(4-10) was markedly reduced (P<0.01), while responses to substance P methyl ester and senktide were only slightly affected. Our results indicate that the physiological contractile responses of the rat duodenum are co-mediated by acetylcholine and tachykinins, for which NK2 receptors seem to be most important.     

Releasing Effect of Calcium and Phosphate on Catechol-amines,ATP, and Protein from Chromaffin Cell Granules

Addition of Ca²⁺3 mM to isolated bovine adrenal niedullary granules incubated in 130 mM K-phosphate buffer pH 6.8–7.2 causes a rapid initial release of noradrenaline (NA), adrenaline (A), ATP and soluble protein. The effect is increased by addition of 1–6 mg/ml of RNA and inhibited by 1–6 mM Mg²⁺. The effect of Ca²⁺is dependent on the presence of phosphate ions in the incubation medium and does not occur in sucrose, RCI or NaCl. Addition of freshly precipitated calcium phosphate is also effective in inducing release, but addition of CaHPO₄ or Ca₂P₂O₇ had no effect. CaCl₂ 0.3–1 mM did not increase the release but was effective together with 1–6 mg/ml of RNA. In the absence of phosphate ions Ca²⁺and Mg²⁺as well as the divalent ions Ba²⁺and Sr²⁺inhibited the release. The catecholamine releasing effect of Ca²⁺in the presence of phosphate is reduced by 2 mM ATP and almost completely prevented by ATP-Mg²⁺. It is suggested that the increased outflow of granular contents is due to membrane alterations induced by Ca-phosphate.     

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 X 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.     

Pre-junctional muscarinic autoreceptors in bovine airways

We searched for pre-junctional inhibitory muscarinic receptors in isolated bovine trachealis strips and bronchial rings. Electric stimulation (ES)-induced tritiated acetylcholine ([³H]-ACh)-release and isometric contractions were determined in muscles incubated with the non-selective muscarinic agonist pilocarpine, the non-selective muscarinic antagonist atropine, the selective M₂-receptor antagonists methoctramine and gallamine, or the selective M₄-receptor antagonist PD102807. Electric field stimulation (EFS)-induced isometric contractile responses were assessed in trachealis strips and bronchial rings treated with 10⁻⁹-10⁻⁵ M methoctramine, gallamine or PD102807. Pilocarpine (10⁻⁶ and 10⁻⁵ M) and atropine (10⁻⁷ M) significantly decreased and increased ES-evoked [³H]-ACh-release, respectively. The enhancing effect of atropine on [³H]-ACh-release prevailed over the inhibitory effect of pilocarpine. M₂- and M₄-receptor antagonists did not increase EFS-induced contraction or ES-induced [³H]-ACh-release. However, 10⁻⁷ M methoctramine, gallamine or PD102807 significantly attenuated the inhibitory effects of pilocarpine 10⁻⁵ M on ES-induced [³H]-ACh-release. Conclusions: Muscarinic autoregulation is present in bovine airways but is not fully accounted for by M₂- and M₄-receptor subtypes.     

Involvement of intracellular calcium ions in the release of the fluorescent dye calcein by cholinergic and α-adrenergic agonists from rat parotid acinar cells

Effects of cholinergic and adrenergic agonists on the secretion of the fluorescent dye calcein were examined to clarify the involvement of calcium ions in the secretion of calcein from acinar cells dispersed from the rat parotid gland. Addition of carbachol (CCh) and noradrenalin (NA), but not isoproterenol (IPR), enhanced the net release of calcein from acinar cells during the subsequent 10 min in a dose range from 10^–8 M to 10^–6 M. The net release of calcein reached a maximum 7 min after the addition of CCh. The release of calcein was suppressed by the simultaneous additions of atropine with CCh, or phenoxybenzamine with NA. Addition of CCh induced a sustained dosedependent increase in the intracellular levels of calcium ions, ([Ca²⁺]_i). Addition of NA at 10^–6 M increased [Ca²⁺]_i. Phenoxybenzamine completely inhibited the NA-induced increase, but propranolol did not. The removal of extracellular calcium ions did not influence the release of calcein induced by 10^–6 M CCh, but it abolished the sustained increase in [Ca²⁺]_i. The transient increase in [Ca²⁺]_i induced by CCh was observed in the absence of extracellular calcium ions. A calcium ion chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) inhibited the CCh-induced release of calcein. The calcium ionophore, A23187 (2.5×10^–6 M), but not 10^–3 M dibutyryl cAMP, evoked the release of calcein. It also increased [Ca²⁺]_i. Removal of extracellular calcium ions suppressed the A23187-induced release of calcein. These results suggest that the release of calcein from parotid acinar cells is transiently induced through an increase in [Ca²⁺]_i by muscarinic and -adrenergic agonists and may represent the initial process of salivary secretion.     

The muscarinic agonist pilocarpine inhibits DNA and interferon-γ synthesis in peripheral blood mononuclear cells

Lymphocyte basal DNA synthesis and proliferative responses to phytohemagglutinin (PHA) showed a dose-dependent (5 × 10⁻⁵ − 5 × 10⁻³ M) inhibition by the muscarinic agonist pilocarpine, in contrast to the basal enhancing effect produced by the M₂ muscarinic-nicotinic agonist carbacol. The effect of pilocarpine was reversed by both atropine (1 × 10⁻⁶ M) and pirenzepine (1 × 10⁻⁷ − 1 × 10⁻⁸ M), M₁ − M₂ and M₁ muscarinic antagonists, respectively. The effect of pilocarpine may thus be specific for the M₁ muscarinic receptor. Pilocarpine also inhibited interferon-gg (IFN-γ)-PHA induced production, but was unable to reverse the pokeweed mitogen (PWM)-induced DNA synthesis. Distinct immunoregulatory activities are suggested for cholinergic muscarinic receptors M₁ and M₂.     

Long term effects of septohippocampal lesions and intrahippocampal grafts on acetylcholine concentration,muscarinic stimulated formation of inositol phospholipids and electrically evoked release of neurotransmitters in the rat hippocampus

Summary Long Evans female rats sustained aspirative lesions of the septohippocampal pathways; subsequently, they received intrahippocampal suspension grafts of fetal septal-diagonal band or hippocampal tissue. The long term (8–10 months post-surgery) effects of these treatments were examined in the hippocampus for the following variables: concentration of hippocampal acetylcholine (ACh), muscarinic-stimulated (carbachol) formation of inositol monophosphate, accumulation of tritiated choline, noradrenaline (³H-NA) and serotonin (³H-5-HT), electrically evoked release of ³H-acetylcholine (³H-ACh), ³H-NA and ³H-5-HT, and choline acetyltransferase (ChAT) activity. The lesions decreased the levels of endogeneous ACh, the accumulation of ³H-choline and ³H-5-HT and the evoked release of both ³H-ACh and ³H-5-HT as well as the ChAT activity, but they failed to significantly affect the muscarinic-stimulated formation of inositol monophosphate and the accumulation and release of ³H-NA. Grafts of hippocampal cells were found to be ineffective on all lesion-induced effects. In contrast, grafts of septal-diagonal band origin attenuated the deficit of hippocampal concentrations of ACh and accumulation of ³H-choline without, however, improving release of ³H-ACh, accumulation and release of ³H-5-HT, and ChAT activity. These observations suggest that: (i) denervation-induced hippocampal muscarinic supersensitivity might not be long-lasting or the lesions, which in some cases spared the lateral edges of the fimbria, failed to induce any muscarinic supersensitivity, (ii) intrahippocampal grafts rich in cholinergic neurons do not foster recovery from the lesion-induced noncholinergic deficits we assessed, (iii) recovery of function may be expressed by some but not all biochemical or pharmacological cholinergic variables and (iv) graft-derived hippocampal reinnervation may be less efficient than the endogenous innervation of intact rats as indicated by the restoration of only some of the variables related to cholinergic function by intrahippocampal septal-diagonal band grafts.     

Stimulation of protein phosphatases as a mechanism of the muscarinic-receptor-mediated inhibition of cardiac L-type Ca2+ channels

Acetylcholine decreases currents through cardiac L-type Ca²⁺ channels after stimulation with agents which elevate levels of cyclic adenosine monophosphate, such as isoproterenol, but there is still a controversy over the mechnisms of this muscarinic effect. We tested the hypothesis of whether, after isoproterenol stimulation, protein phosphatases are activated by acetylcholine. Whole-cell currents were recorded from guinea-pig ventricular myocytes. The effect of 10^–5 M acetylcholine on currents induced by 10^–8 M isoproterenol was studied in the absence or presence of protein phosphatase inhibitors. Three agents reduced the acetylcholine response: okadaic acid (3 or 9 · 10^–6 M) and cantharidin (3 · 10^–6 M) added to the pipette solution, and bath-applied fluoride (3 mM). In contrast, pipette application of other phosphatase inhibitors, namely the inhibitor PPI₂ (1000 U/ml), ciclosporin (10^–5 M), or calyculin A (10^–6 M) did not significantly diminish the acetylcholine effect. Interestingly, there was no correlation between the effects of the compounds on basal Ca²⁺ current and their interference with the muscarinic response. An activation of type 2A phosphatases by acetylcholine would explain these findings. Indeed, okadaic acid is 3 orders of magnitude more potent in vitro in its inhibition of this isoform (purified from cardiac myocytes) than is calyculin A, while type-1 phosphatases are inhibited equally. The data support the attractive possibility that stimulation of protein phosphatases is part of the signal transduction cascade of Ca²⁺ channel inhibition by acetyl-choline.     

Effects of κ- and μ-opioid agonists on cholinergic neurotransmission and contraction in isolated bovine trachealis

The effects of the selective μ-opioid agonist DAMGO and the selective κ-opioid agonist U-50488H on tritiated acetylcholine release ([³H]-ACh) and contractile responses to electrical stimulation (ES) were simultaneously determined in isolated bovine trachealis. The inhibitory effect of DAMGO 10⁻⁵ M on [³H]-ACh release was not significantly different from the effect of the non-selective muscarinic agonist pilocarpine 10⁻⁵ M, whereas the effect of U-50488H 10⁻⁵ M was significantly greater. The effects of both opioids were not significantly different when muscles were pre- or co-incubated with the unselective muscarinic antagonist atropine 10⁻⁷ M. Both DAMGO and U-50488H attenuated ES-induced contraction and this effect was significantly correlated with the inhibition of [³H]ACh-release (r² = 0.8552). These data suggest that (1) opioids are important modulators of airway smooth muscle tone, (2) their effect is not altered by the activity of muscarinic autoregulation, and (3) their inhibitory effect of airway smooth muscle contraction can be almost totally explained by inhibition of ACh release.     

Muscarinic receptors mediate direct inhibition of GABA release from rat striatal nerve terminals

The effects of acetylcholine (ACh) on the depolarization-evoked release of [3H]gamma-aminobutyric acid ([3H]GABA) have been investigated using synaptosomes prepared from rat corpus striatum and depolarized by superfusion with 9 mM KCl. Acetylcholine inhibited the [3H]GABA overflow in a concentration-dependent manner. The maximal effect was about 50%. The IC50 value (concentration producing half-maximal effect) amounted to 1 microM, in the absence of acetylcholinesterase inhibitors. The effect of ACh on the K(+)-evoked [3H]GABA release was counteracted by the muscarinic receptor antagonist atropine, but not by the nicotinic receptor antagonist mecamylamine or by the selective M1 antagonist pirenzepine. The data show that muscarinic receptors with low affinity for pirenzepine are localized on GABAergic nerve endings in rat corpus striatum where they may directly inhibit the release of GABA.     

Further studies on properties of renin granules isolated from rat kidney cortex

The properties of renin granules isolated from rat renal cortex were studied. Renin granules were thermolabile since in 10 min at 0°C twice as much renin was released as at +37°C. Addition of Ca++ (10^-6 M-10^-2 M) did not affect the spontaneous release at +37°C, pH 6.5, during 10 or 30 min incubation. However, when pH was elevated to above 7, renin release was significantly increased by Ca++ (10^-3 M). Additions of various amounts of KCl, NaCl or MgCl₂, which increased the osmolality less than 20 mOsm/kg, did not affect the stability of the renin granules. Mg-ATP (0.5 and 5 mM) as well as Mg-GTP (5 mM) stabilized renin granules at +37°C, pH 6.5, but the corresponding nitrogen analogues Mg-AMP-PNP and Mg-GMP-PNP (0.5 and 5 mM) were not effective. Neither did Mg-AMP (5 mM) nor ATP (5 mM) without Mg++ affect the renin release. No stabilization was observed by Mg-ATP and Mg-GTP in the purified granule preparations. The results suggest the importance of the cleavage of the terminal phosphate in the stabilization process. When the granules prepared at 300 mOsm/kg were first kept at hyperosmotic medium (range 300–1650 mOsm/kg) and then moved back to 300 mOsm/kg, the granules tend to lyse the more the greater was the reduction of the osmolality. The granules were more stable in isotonic sucrose than in isotonic ionic medium.     

Presynaptic autoinhibition during rest and sodium-pump inhibition in isolated rat portal vein preparation

In the presence of cocaine and corticosterone low-frequency (2 Hz) nerve stimulation evoked release of [3H]noradrenaline measured from isolated rat portal vein preparation. In normal Krebs solution exogenously applied l-noradrenaline (3 X 10(-8)-10(-6) M) significantly reduced the nerve-evoked [3H]noradrenaline release. The IC50 value of L-noradrenaline proved to be 1.8 X 10(-7) M. Yohimbine (3 X 10(-7) M) maximally blocked the alpha 2-adrenoceptors and enhanced nerve-evoked [3H]noradrenaline release. In the presence of 5.9 mM external K+, ouabain up to 10(-4) M did not affect either the resting or the stimulation-evoked release of radioactivity from tissues. In the absence of external K+ both the resting and the nerve-evoked release of [3H]noradrenaline increased markedly. When K+ was readmitted to preparations which had been kept in K+-free solution both the resting and the stimulation-evoked [3H]noradrenaline release were greatly reduced temporarily. In K+-free solution L-noradrenaline (10(-6) M) and yohimbine (3 X 10(-7) M) failed to significantly alter the nerve-evoked release. However, 3 X 10(-6) M yohimbine in K+-free solution significantly increased the stimulation-evoked release of [3H]noradrenaline. It is concluded that presynaptic alpha 2-adrenoceptor-mediated "negative feed-back" is present in rat portal vein preparations which can be inhibited by the preferential alpha 2-adrenoceptor blocker, yohimbine. However, if the Na+-pump is inhibited (which by itself enhanced the transmitter release), presynaptic autoinhibition is more pronounced, since a high concentration of yohimbine is required to block it.     

Presynaptic effect of L-glutamic acid on the release of dopamine in rat striatal slices

The release of [³H] dopamine ([³H] DA) was estimated in serial superfusate fractions of rat striatal slices continuously superfused with L-[3,5-³H]-tyrosine. L-glutamic acid (5 · 10^?5 M), but not the D-stereoisomer, increased the spontaneous release of [³H] DA (60%). The stimulating effect of L-glutamic acid was still observed in the presence of tetrodotoxin (5 · 10^?7 M), suggesting that the amino-acid acts at a presynaptic site. Moreover, atropine (10^?6 M) or pempidine (10^?5 M) which blocks the acetylcholine (ACh) evoked release of [³H] DA did not reduce the stimulatory effect of L-glutamic acid on [³H] DA release, thus excluding the possible intervention of striatal cholinergic neurons. The data obtained support the hypothesis of a direct control of DA release from nerve terminals by glutamatergic neurons.     

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 [Ca²⁺] 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 [Ca²⁺] concentration. In 0.1 mM [Ca²⁺] overflow of [³H]noradrenaline was completely blocked. However, addition of 30 mM tetraethylammonium to 0.1 mM [Ca²⁺]-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 [Ca²⁺] plus phentolamine) to the same extent as with tetraethylammonium and low [Ca²⁺], 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 [Ca²⁺] for the release process.     

Estrogen inhibition of noradrenaline release in the rabbit oviduct1

In the present study, we investigated the influence of estrogen on ³H-noradrenaline (³H-NA) release induced in the oviductal isthmus by electrical stimulation, potassium and calcium. The fractional release of ³H-NA was measured in oviducts isolated from ovariectomized rabbits and from ovariectomized rabbits treated with estradiol cypionate, 70 μg/kg im 72 h before an experiment. Electrical field stimulation of the intramural nerves induced muscle contraction and augmented the release of labelled NA from the muscle. The ³H-NA release was reduced after estrogen treatment when reuptake of NA into the nerve terminals was blocked by desipramine, 10^-6 M. Estrogen also reduced the ³H-NA release evoked by exposure of the oviducts to 121 mM KCI in the presence of calcium (2.5 mM) and in a high potassium, calcium-free medium upon the addition of 2.5 mM calcium. In the presence of desipramine a small fraction of ³H-NA was released in high potassium, calcium-free medium. This release was unaffected by estrogen. These results suggest that estrogen reduces the release of NA from the adrenergic nerves within the oviduct and that this action is exerted primarily on the calcium-dependent release. It therefore might be due to a reduction in the entry of calcium into the nerve terminal.     

The effect of preganglionic nerve stimulation on the accumulation of certain analogues of choline by a sympathetic ganglion.

1. Cat superior cervical ganglia were perfused with a Krebs solution containing 10(-6) M [3H]homocholine (2-hydroxypropyl-trimethylammonium) or 10(-5) M [14C]triethylcholine (2-hydroxyethyl-triethylammonium). Preganglionic nerve stimulation (20 Hz) increased the accumulation of homocholine (3-2-fold) and of triethylcholine (2-1-fold). This increased accumulation during stimulation was not the result of increased metabolism. 2. The increased accumulation of homocholine or triethylcholine induced by pregnaglionic nerve stimulation was not reduced by tubocurarine or by atropine, but it was blocked by choline and by hemicholinium. These results suggested that preganglionic nerve stimulation increased choline analogue accumulation into cholinergic nerve terminals. 3. The increased accumulation of homocholine or of triethylcholine induced by preganglionic nerve stimulation was reduced when the Ca2+ concentration was reduced and was abolished in the absence of Ca2+. However, changes in the Mg2+ concentration which depressed acetylcholine (ACh) release by amounts comparable to those induced by altered Ca2+ concentrations did not alter the uptake of homocholine or triethylcholine. It is concluded that the uptake of choline analogues is not regulated by transmitter release but that stimulation increases the uptake of the choline analogues by a Ca2+-dependent mechanism. 4. The accumulation of ACh by ganglia perfused with a Krebs solution containing choline and high MgSO4 (18 mM) was measured. The ACh content of these ganglia did not increase, although choline transport presumably exceeded that necessary for ACh synthesis to replace released ACh. It is concluded that choline transport does not limit ACh synthesis in ganglia.     

Effects of aging on rat cortical presynaptic cholinergic processes

We studied the effects of aging on the [³H]-choline uptake, acetylation, [³H]-ACh release and muscarinic modulation of [³H]-ACh release in cortical synaptosomes prepared from Fischer 344 male rats. Our results indicate that 6 and 24 month old rats take up and acetylate [³H]-choline to a similar extent, but that the older animals release significantly less [³H]-ACh in response to K⁺-depolarization than the young adults do. This difference in K⁺-induced release is not due to a difference in presynaptic muscarinic receptor inhibitory activity since the older animals appear to be, if anything, slightly less sensitive to oxotremorine than the younger animals are. Atropine (1 μM) had no effect on ACh-release but blocked oxotremorine-induced modulation. Our results suggest that acetylcholine release is decreased in synaptosomes prepared from old rats although the presynaptic muscarinic regulation of release is functional. Thus, muscarinic receptor-mediated release-modulation is a potential site for pharmacologically altering ACh release.