Stretch and anesthetic dependency of atrial natriuretic peptide release demonstrated by an ultrastructural assay

Abstract

Using an ultrastructural assay developed to quantify the secretion of atrial natriuretic peptide-containing granules, release of the hormone, in response to different degrees of atrial distension, is directly demonstrated at the cellular level. The ultrastructural assay developed uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial granules in the anesthetized rat. Secretory granules, which retain the capacity to undergo exocytosis throughout a 30-minute tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane, with the core contents retained. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different stimuli to be assessed. By altering the height of the perfusate, perfusion pressure and hence the degree of distension of the right atrium can be increased, and this causes a proportional rise in the release of secretory granules from individual myocytes. An anesthetic regime incorporating fentanyl citrate was found to increase significantly the rate of granule release, and this was further augmented by atrial distension. Quantification of the numbers of cytoplasmic granules under the same conditions did not reveal a reduction in granules. This is thought to be because only a small pool of granules is recruited for exocytosis, and granule production may continue during the perfusion period. Our assay of atrial secretory granule release allows the effect of a variety of stimulatory and inhibitory agents to be assessed directly at the cellular level and provides an independent comparison with previous biochemical data from whole animal and isolated organ studies.

Effect of neuromimetics upon the release of atrial natriuretic peptide granules: Are multiple pathways involved in secretion?

“…Control atrial myocytes, fixed after 15 min or 30 min tannic acid perfusion, showed a pattern of granule release similar to that seen previously in Sagatal-anaesthetised rats (Newman and Severs, 1993). There was a significant increase in the numbers of arrested granules found with time.…”

“…The mechanism they suggested involved the budding of clathrin-coated vesicles from immature secretory granules containing inefficiently aggregated secretory proteins-vesicles that could be secreted in the absence of stimulation. We have reported the occurrence of such coated immature granules in atrial tissue (Newman and Severs, 1990)) and similar coats also appear to be involved in the retrieval of granule membrane after fusion with the plasma membrane (Newman and Severs, 1993). However, when budding from cytoplasmic granules, not all vesicles appear to contain granule contents, and, additionally, the number of vesicles formed in this manner appears to be small.…”

“…By directly assaying release of the ANP-containing secretory granules after neuromimetic application, we have sought to provide a fresh insight into the mechanisms involved. We have previously reported arrested exocytosis of these granules, labelled with anti-ANP antibodies (Newman et al, 1991), and shown that exocytosis increases with atrial distension, the main stimulus to release, and with an anaesthetic regime incorporating the opioid agonist fentanyl citrate (Newman and Severs, 1993). The results presented here, however, indicate that, of the neuromimetics used, phenylephrine and isoproterenol do not significantly alter the release rate of secretory granules, at least in the 30 min period after drug application, whereas acetylcholine causes a distinct reduction in release.…”

“…This technique, in situ tannic acid perfusion, allows ANP release to be quantified at the most fundamental level, that of the exocytosing 0 1996 WILEY-LISS, INC ANP-containing secretory granules (Newman and Severs, 1992; Newman et al, 19911, which is not possible using conventional electron microscopy (Newman and Severs, 1990). Using tannic acid perfusion, we have previously shown that it is possible to correlate secretory granule release with the degree of atrial distension, and we have also investigated the effects of different anaesthetics demonstrating the stimulatory role of opioids in granule release (Newman and Severs, 1993).In the present study, we have investigated the effect of neuromimetics (isoprenaline, phenylephnne, and acetylcholine) upon secretory granule release, using tannic acid perfusion to arrest secretion. The results are interpreted in relation to previous studies, and a new model for the storage and release of the hormone is proposed to clarify previous findings.…”