Role of the alpha-adrenoceptor in regulating noradrenaline overflow by nerve stimulation. 1971

Abstract

1. A study of the actions of phenoxybenzamine on transmitter overflow, neuronal and extraneuronal uptake of noradrenaline and in causing α-adrenoceptor blockade was carried out using the isolated cat nictitating membrane preparation.

2. Phenoxybenzamine increased transmitter overflow elicited by nerve-stimulation at 10 Hz in a concentration dependent manner in the range 10⁻⁸ to 10⁻⁵ g/ml.

3. Neuronal uptake of [³H]-noradrenaline was not inhibited by concentrations lower than 10⁻⁶ g/ml of phenoxybenzamine. With 10⁻⁷ g/ml of phenoxybenzamine a significant increase in transmitter overflow was obtained, although neuronal uptake of noradrenaline was not affected. Higher concentrations of phenoxybenzamine (10⁻⁶ and 10⁻⁵ g/ml) inhibited the neuronal uptake of noradrenaline and further increased transmitter overflow.

4. Extraneuronal uptake of [³H]-noradrenaline was inhibited only with the highest concentration of phenoxybenzamine tested (10⁻⁵ g/ml) and therefore appears to be unrelated to the effects on transmitter overflow.

5. There was a significant correlation between the degree of α-adrenoceptor block produced by phenoxybenzamine and the increase in transmitter overflow obtained by nerve stimulation.

6. These results indicate that phenoxybenzamine, in addition to increasing overflow by preventing reuptake of noradrenaline, may increase transmitter release.

7. The possibility that phenoxybenzamine acts on α-adrenoceptors in the adrenergic nerve terminal is discussed. These receptors would be involved in a negative feedback mechanism regulating transmitter release.

Fig. 1

Effects of phenoxybenzamine on dose-response curves to noradrenaline in the normal nictitating membrane of the cat. Ordinate: development of tension in percent of the maximum. Abscissa: concentration of (—)-noradrenaline (NA). •—•, Controls; ×—×, phenoxybenzamine 10⁻⁸ g/ml; ○—○. phenoxybenzamine 10⁻⁷ g/ml; δ—δ, phenoxybenzamine 10⁻⁶ g/ml; □—□. phenoxybenzamine 10⁻⁵ g/ml. Values are expressed as means ± s.e. of the mean.

Fig. 2

Correlation between the accumulation of [³H]-noradrenaline and the endogenous noradrenaline concentration in normal muscles of the nictitating membrane. Abscissa: endogenous noradrenaline concentration of the nictitating membrane (μg NA/g). Ordinate: accumulation of [³H]-noradrenaline (ng ³H-NA/g); r = 0·67; n = 18; b = 20·7 ± 5·7; P<001.

Fig. 3

Overflow of radioactive products elicited by nerve stimulation. Ordinate: nCi per 100 mg of tissue. The bars beneath the abscissa indicate the 5 min period of stimulation. The open columns represent the spontaneous outflow in consecutive 5 mm samples obtained 90 min after the end of the incubation with [³H]-noradrenaline. NA, noradrenaline; CDA, catecholdeaminated metabolites; NMN, normetanephrine; OMDA, O-methylated-deaminated metabolites. The black portion is intended to represent the increased overflow produced by nerve stimulation. Nerve stimulation: 10 Hz, 0·5 ms duration and supramaximal voltage. The values shown are the means of four experiments.

Fig. 4

Correlation between the radioactivity in the tissue and the overflow elicited by nerve stimulation. Filled circles are the results obtained in response to the first period of stimulation in individual experiments. The open circles identify the individual values in response to second stimulations in experiments in which two consecutive stimulations were performed. The numbers indicate the four experiments in which two stimulation periods were used. r = 0·556; b = 1·44 ± 0·44; n = 25; P<001. Only the filled circles were used for the calculation of the regression.

Fig. 5

Responses of the medial muscle of untreated nictitating membranes to two successive 5 min periods of stimulation (10 Hz, 0·5 ms, supramaximal voltage). The responses plotted are mean ± s.E. of the mean. (n = 4.) a, First stimulation; b, second stimulation.

Fig. 6

Responses of the medial muscle of the nictitating membrane to a 5 min period of nerve stimulation (10 Hz, 0·5 ms; supramaximal voltage). The responses plotted correspond to the mean ± s.e. of the mean, a, Controls (n = 25); b, phenoxybenzamine 10⁻⁸ g/ml (n = 6); c, phenoxybenzamine 10⁻⁷ g/ml (n = 7); d, phenoxybenzamine 10⁻⁶ g/ml (n = 6); c, phenoxybenzamine 10⁻⁵ g/ml (n = 6).

Fig. 7

Comparison of the effects of phenoxybenzamine on neuronal accumulation of [³H]-noradrenaline and on the overflow of transmitter elicited by nerve stimulation. ○—○, Fractional release per shock (× 10⁻⁵); ▴—▴, neuronal accumulation of [³H]-noradrenaline (ng [³H]-noradrenaline per μg of endogenous noradrenaline). Values are mean ± s.e. of the mean. *P<0·05; ** P<0·01.

Fig. 8

Correlation between block of responses to nerve stimulation and transmitter overflow in the presence of phenoxybenzamine. Each point represents an individual experiment. Abscissa: ratio of the responses to nerve stimulation in the presence and in the absence of of phenoxybenzamine. Ordinate: fraction release per shock (×10⁻⁵) induced by a 5 min period of nerve stimulation (10 Hz, 0·5 ms, supramaximal voltage). The controls were not mcluded in the correlation and are shown as mean ± s.e. ○, Control; •, phenoxybenzamine, 10⁻⁸ g/ml; δ, phenoxybenzamine, 10⁻⁷ g/ml; □. phenoxybenzamine, 10⁻⁶ g/ml; ▴, phenoxybenzamine, 10⁻⁵ g/ml. r = 0·63, b = 4·19 ± 1·06, n = 25, P<0·01.