Muscarinic inhibition of acetylcholine release from a novel in vitro preparation of the guinea-pig trachea.

An isolated preparation of the guinea-pig trachea is described which allows the simultaneous measurement of acetylcholine release and smooth muscle contraction. Incubation of the epithelium-free preparation with [3H]choline resulted in the formation of [3H]acetylcholine. Electrical stimulation caused the release of [3H]acetylcholine and a contractile response. Tetrodotoxin and omission of calcium from the medium abolished both the evoked release and contractions. The muscarinic agonists oxotremorine, carbachol and pilocarpine concentration-dependently inhibited the electrically evoked acetylcholine release and contracted the tracheal smooth muscle. Pre- and postsynaptic EC50 values for a given agonist were not different. Atropine (100 nmol/l) significantly facilitated the evoked acetylcholine release. A concentration of 10 nmol/l atropine did not change the evoked release but antagonized the inhibitory effect of oxotremorine. It is concluded that presynaptic muscarine autoreceptors inhibit the release of acetylcholine from parasympathetic nerves of the guinea-pig trachea.

Prejunctional effects of muscarinic agonists on 3H-acetylcholine release in the rat urinary bladder strip.

The inhibitory effects of some muscarinic agonists on tritiated acetylcholine release evoked by field stimulation were investigated in the rat urinary bladder strip. The acetylcholine stores of the preparation were labelled with 3H-choline. Electrical field stimulation caused an outflow of tritium, reflecting the release of 3H-acetylcholine. The release of 3H-acetylcholine was decreased in a concentration-dependent manner by all the agonists tested: oxotremorine, muscarone, muscarine, carbachol and methylfurtrethonium. On the contrary, only muscarine and muscarone enhanced the basal efflux of tritium in a concentration-dependent fashion. Concentration-response curves were determined both at 2 Hz and at 1 Hz by using intermittent administration of the drugs. Maximal depression in release (by 78-82%) was observed in experiments at 1 Hz. A similar inhibition was obtained at 2 Hz frequency only when a low concentration of calcium (0.6 mM) in the medium was used. Oxotremorine was the most potent among the tested compounds with the same intrinsic activity as the other drugs. In contrast to the other agonists investigated, oxotremorine showed in about 10-fold greater potency at pre- than at postjunctional muscarine receptors in the rat urinary bladder. This difference might depend either on heterogeneity of muscarine receptors or on different mechanism(s) relating to the transducing properties of receptors at the pre- and postjunctional level. A comparison between the relative prejunctional potencies in the rat urinary bladder and in the guinea pig myenteric plexus (data from the literature) suggests that prejunctional muscarine receptors are similar in these tissues. Furthermore, the findings obtained with a low concentration of calcium in the medium may support the view that intraneuronal availability of calcium plays a significant role in modulating the prejunctional negative feed-back mechanism in the rat urinary bladder.

Formation and release of [3H]acetylcholine in the rat urinary bladder strip.

The relationship between different frequencies of loading stimulation and [3H]acetylcholine (ACh) formation and release from nerve terminals has been investigated in extratrigonal strips of the urinary bladder of the rat. An increase in frequency (0.2, 0.4 and 0.8 Hz) for the 30 min incubations with [3H]choline produced an enhancement of storage of [3H]ACh from 19.5 to 34% of total tritium content in the tissue. Higher frequencies (1.6 and 3.2 Hz) failed to increase storage further on. The [3H]choline content did not vary significantly. Electrical field stimulation at 2 Hz (360 shocks) produced a release of tritium. The evoked outflow was higher when the strip was loaded at 0.8 Hz than at the other frequencies tested. Both [3H]ACh and [3H]choline were measured in the perfusate of strips preloaded at 0.8 Hz. Most of the induced outflow was found to be [3H]ACh, as in previous experiments carried out using 0.2 Hz as a loading frequency. The findings suggest that in the rat urinary bladder strip loading at 0.8 Hz is suitable for increasing the formation and the resulting release of [3H]ACh during electrical stimulation.

Presynaptic inhibitory muscarinic receptors modulating [3H] acetylcholine release in the rat urinary bladder.

The occurrence of presynaptic muscarinic receptors inhibiting the release of acetylcholine (ACh) from nerve terminals was investigated in the rat urinary bladder. Strips from the extratrigonal area were preincubated with [3H]choline and stimulated at 0.2 Hz. Both [3H]ACh and [3H]choline content were measured in the tissue. The uptake of tritiated choline was prevented by hemicholinium-3. The field stimulation at 2 Hz (360 shocks) produced a release of tritium. Most of the induced outflow was found to be [3H]ACh. Both tetrodotoxin treatment and calcium omission from the medium prevented such an evoked-outflow of tritium. When two electrical stimulations (S1 and S2) at 2 Hz (360 shocks) were carried out at 45-min intervals, an S2/S1 ratio of 0.82 was found. Physostigmine reduced the evoked-release of [3H]ACh whereas atropine increased it in a concentration-dependent manner. Atropine antagonized the inhibitory effect of physostigmine, so that the S2/S1 ratio did not vary significantly from control experiments. Both carbachol and muscarine strongly decreased the [3H]ACh evoked outflow. Muscarine increased the spontaneous outflow of tritium also. These findings suggest that the urinary bladder of the rat is equipped with presynaptic inhibitory muscarinic receptors modulating ACh release from cholinergic postganglionic neurons.

Analysis of the relaxing effect of dopamine on the isolated rat jejunum.

The relaxing effect of dopamine on the rat isolated jejunum has been studied. Dopamine was found 170 times less potent than noradrenaline and 3 times more potent than tyramine. The relaxing effect of dopamine does not show the tachyphylaxis phenomenon, is present in preparations from rats pretreated with reserpine and is not influenced by cocaine. These results indicate that dopamine has a direct action. To characterize the receptor(s) through which dopamine causes intestinal relaxation the alpha-blocker phentolamine, the beta-blocker propranolol and the dopamine receptor blockers haloperidol and cis-alpha-flupenthixol, alone or in combination have been tested. 40% of inhibition of the response to dopamine was obtained with phentolamine, 25% with propranolol and 30% with haloperidol or cis-alpha-flupenthixol. Combining together three antagonists acting on three different receptors it was possible to obtain 70% of inhibition of the responses to dopamine. It is concluded that alpha and beta adrenoceptors and specific dopamine receptors are involved in the direct relaxing action of dopamine.

Uptake and intracellular distribution of lanthanum.

The present investigation of the cellular distribution of lanthanum was undertaken in order to control the validity of the "Lanthanum method" used for the study of the cell calcium compartments. The presence of lanthanum was evaluated in the isolated guinea-pig heart and its subcellular fractions perfused with a lanthanum-containing Tyrode solution. Lanthanum was determined by instrumental neutron activation analysis. Under the adopted experimental conditions (30-min incubation in the presence of 12.5 microM lanthanum), lanthanum is carried across the cell membrane and is taken up by subcellular organelles. These results confirm the limited validity of investigations based on of the "Lanthanum method".

Effect of lanthanum on the turnover of calcium in rat uterus.

Previous investigations suggest that lanthanum might enter uterine smooth muscle cells and work as intracellular calcium displacing agent. The present investigation had been carried out in order to confirm if lanthanum develops an intracellular effect. Experiments show that lanthanum brings about a marked increase of the intracellular calcium; the comparison of the uptake and of the wash-out curve of 45Ca shows that lanthanum induces a lowering of the rapid phase of 45Ca release from rat uterus, while the uptake of the labelled ion is not modified or is even enhanced. The present data demonstrate that the action of lanthanum in rat uterus is limited to the cell membrane, whose calcium extruding properties are inhibited.

Experimental investigations on the contraction induced by lead in arterial smooth muscle.

Previous observations suggested that the turnover of tissue calcium might be involved in the mechanism of smooth muscle contraction induced by lead. In the present investigations, the effect of lead on calcium exchangeability has been studied in the isolated rat tail artery. Experimental results suggest that the mechanism of lead action might be identified with a tissue calcium accumulation and with a lead-to-calcium competition. Evidence exists that the site of action is located in the cell membrane, where lead inhibits the processes of calcium extrusion, and in the intracellular calcium stores, whose calcium binding capacity is lowered; both processes induce an increase of the cellular exchangeable calcium available for contraction.

Microbiologic and pharmacologic properties of a new ampicillin derivative: Ampicillin-guaiacolsulfonate.

Ampicillin-guaiacolsulfonate, a new derivative of ampicillin, preserves the antibacterial properties of ampicillin, is suitable for infection owing to its good water solubility and it has far higher stability in dry state than has sodium ampicillin. Ampicillin-guaiacolsulfonate is better orally absorbed in rabbit than is ampicillin. In vitro experiments carried out with the model system by Rosano and Schulman, and measurements of the intestinal absorption in the everted sac jejunal preparation, carried out with 14C-labelled compounds, demonstrate that the new ampicillin derivative is better absorbed by the intestine because of an increase in the intestinal permeability of the molecule itself.