Segregation of nitric oxide synthase expression and calcium response to nitric oxide in adrenergic and noradrenergic bovine chromaffin cells

Abstract

Previous work has demonstrated that nitric oxide can be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells. Since standard chromaffin cell cultures are mixed populations of noradrenaline and adrenaline producing cells, it would seem important to understand the functional differences between these individual components. The use of fluorescence imaging techniques for the recording of cytosolic calcium from single chromaffn cells together with the immunoidentification of individual cells with specific antibodies against tyrosine hydroxylase, N-phenyl ethanolamine methyl transferase and nitric oxide synthase, has allowed us to measure single-cell calcium responses in identified adrenergic, noradrenergic and nitrergic chromaffin cells, thus helping us to clarify the differential role of nitric oxide in the function of these chromaffin cell types. 53±2% of chromaffin cells were able to synthesize nitric oxide (nitric oxidesynthase-positive cells), these cells being mainly noradrenergic (82±2%). Results indicate that nitric oxide donors such as sodium nitroprusside, molsidomine and isosorbide dinitrate evoke [Ca²⁺]_i increases in a 62±4% of chromaffin cells, the response to nitric oxide donors being between 30 and 50% of that of 20 μM nicotine. Cells responding to nitric oxide donors were mainly adrenergic (68±5%) although 45±9% of noradrenergic cells also gave [Ca²⁺]_i increasing responses. The distribution of nitric oxide responding cells between nitric oxide synthase-positive and negative was very similar in the whole population (63 ± 5 and 60 ± 7%, respectively), but these differences were more prominent when considering the distribution of nitric oxide response between noradrenergic and adrenergic nitric oxide synthase-positive cells; while 73±6% of adrenergic nitric oxide synthase-positive cells evoke [Ca²⁺]_i increases by nitric oxide stimulation, only 35 ± 11% of noradrenergic nitric oxide synthase-positive cells respond.

Taken together these results seem to indicate that (i) nitric oxide could act within adrenal medulla as both an intracellular and intercellular messenger; and (ii) noradrenergic cells seem to be specialized in nitric oxide synthesis while adrenergic cells with an endocrine function could mainly act as a target of neurosecretory action of this second messenger.