Atrial-specific granules in situ have high calcium content, are acidic, and maintain anion gradients.

Abstract

The composition and pH of atrial-specific granules of rat heart were determined by electron probe x-ray microanalysis and fluorescence microscopy, respectively. The high (75 mmol/kg of dry weight) calcium content and higher than cytoplasmic concentration of chloride in atrial-specific granules were visualized in high-resolution x-ray maps. The C1-content of granules and cytoplasm decreased and a bromide gradient (granule > cytoplasm) was established during incubation in low-chloride, NaBr-containing solutions.Scanning confocal fluorescence light microscopy of live atria incubated with acridine orange demonstrated dye accumulation, indicative of low intragranular pH. We conclude that the granules represent a hitherto unrecognized intracellular store of cardiac calcium and can develop and maintain an anion gradient, presumably through cotransport by means of a proton-pumping ATPase.Atrial-specific granules (ASGs), described nearly three decades ago and present in the heart of most vertebrates (1, 2), contain precursors of a family of vasodilator and natriuretic polypeptides, collectively called atrial natriuretic factor (ANF) (3-7). The heart can function as an endocrine organ, releasing ANF in response to changes in atrial distending pressure and other stimuli (8). We now show that ASGs share other properties of endocrine secretory granules, such as an acidic interior and the ability to establish a sizable anion gradient. We also show that these organelles have a high Ca content and are a hitherto unknown store of intracellular Ca in atrial myocardium. Based on the known sequences ofANF prohormone (9, 10) and the Ca-binding protein calsequestrin (11), we suggest that the high Ca content of ASGs is probably due to binding to grouped acidic residues in the aminoterminal sequence of the prohormone. METHODS AND MATERIALSMale Wistar rats (250-300 g of body weight) were killed by cervical dislocation, and the hearts were quickly excised and placed in a well-oxygenated modified Krebs/bicarbonate solution (pH 7.4) at 32°C containing (mM) NaCl, 125; KCI, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 18.7; CaCl2, 1.2; dextrose, 5.6. The right atrium was everted over a 1.5-mm (diameter) silver pin and tied at the base. Tension was not monitored. The atria in Krebs solution generally beat spontaneously up to the point of freezing in either systole or diastole. To determine whether ASGs can establish an anion gradient de novo, atria were incubated for 15 min at 32°C in a solution (pH 7.4) containing (mM) sodium methanesulfonate, 105; NaBr, 20; potassium methanesulfonate, 4.7;

Atrial Specific Granules. An Intracellular Calcium Store.

“…In the present work, it was observed that the ASG contained high Ca content up to 81 mmol/kg dry wt., which approximates the value reported by Somlyo [18], and that Ca2+-ATPase was located on the membrane of ASG. Based on these findings, it is postulaled that the Ca2+-ATPase on the membrane of ASG may pump Ca2+ from cytosol (low Ca2+ concentration side) into ASG (high Ca2+ side) and, therefore, play a part in maintaining the high Ca concentration in the ASG.…”

“…Somlyo et al [18] reported a high Ca concentration in ASG and assumed it to be an ICaS. However, he did not explore how the high Ca concentration was maintained and whether the Ca stored in ASG participated in the ANP secretion.…”

Polyphosphate Is a Novel Pro-inflammatory Regulator of Mast Cells and Is Located in Acidocalcisomes

“…Our results further support the concept that acidocalcisomes are lysosome-related organelles and indicate that acidocalcisomes and serotonin-containing mast cell granules share several properties: they are acidic due to the operation of proton pumps and able to accumulate acidophilic dyes such as acridine orange; they can store large amounts of calcium and other cations such as potassium, sodium, magnesium, and zinc; they can release Ca 2ϩ in the presence of alkalinizing agents that also promote the hydrolysis of polyP; they contain large amounts of phosphorus in the form of polyP; and they have very high density when observed by direct electron microscopy. Other secretory granules, such as those in pancreatic islets of Langerhans (46), atrial specific granules (47), and secretory chromaffin granules (48) are known to possess large amounts of phosphorus and cations, as…”

Ca2+ Content and Expression of an Acidocalcisomal Calcium Pump Are Elevated in Intracellular Forms ofTrypanosoma cruzi

“…In mammalian cells, Ca 2ϩ has also been reported to be present in acidic organelles carrying vacuolar-type proton pumps, such as endosomes, lysosomes, and the trans-Golgi network, and secretory granules such as chromaffin, pancreatic zymogen, and atrium-specific granules (19,37,50,61), but the functional significance of the high Ca 2ϩ content of these organelles is unknown (37). Recent evidence has indicated, however, that second messengers such as inositol 1,4,5-trisphosphate and cyclic ADP ribose can release Ca 2ϩ from pancreatic zymogen granules (19), although this conclusion is disputed by other investigators (57).…”