Abstract
A novel combination of two single cell assays allowed the simultaneous measurement of intracellular calcium concentration and hormone secretion in normal pituitary cells. [Ca2+]i was recorded using the fluorescent Ca2+ indicator fura-2 and digital imaging microscopy. This technique was combined with a reverse hemolytic plaque assay for growth hormone in order to identify somatotropes and quantitate the amount of hormone released. A dynamic profile of rhythmic calcium oscillations was found in spontaneously secreting somatotropes. Each somatotrope displayed a distinct frequency (one pulse every 5-30 s) and amplitude (range 50-450 nM) generated asynchronously from cell to cell. The amount of growth hormone (GH) released correlated directly with both the frequency and amplitude of calcium oscillations at the level of single GH cells. Furthermore, calcium excursions in somatotropes were rapidly suppressed by either (i) removal of extracellular calcium, (ii) somatostatin (1 mM), or (iii) the calcium channel blockers cobalt (2 mM) and verapamil (100 microM). These observations demonstrate that spontaneous calcium oscillations are characteristic for normal somatotropes. These oscillations are related to spontaneous hormone secretion and due to influx through calcium channels in the membrane. Somatostatin, the physiologic inhibitor of GH secretion, suppresses calcium transients. These findings suggest that the intracellular signaling information may be encoded both in the frequency and amplitude of calcium oscillations.
Highlights
Spontaneous Oscillationsof Intracellular Calcium and Growth Hormone Secretion*[Ca2+]1was recorded using the fluorescent Ca2+indicator fura-2 and digital imaging microscopy
A novel combinationof two singlecell assays allowed the simultaneousmeasurementof intracellular calcium concentration and hormone secretion in normal pituitary cells. [Ca2+]1was recorded using the fluorescent Ca2+indicator fura-2 and digital imaging microscopy
Two subpopulations exist among somatotropes. 29%of all pituitary cells spontaneously secreted growth hormone (GH)
Summary
[Ca2+]1was recorded using the fluorescent Ca2+indicator fura-2 and digital imaging microscopy This technique was combined with a reverse hemolytic plaque assay for growth hormone in order to identify somatotropes and quantitate the amount of hormone released. In order to investigate the role of calcium in normal somatotropes, calcium ions and hormone release were measured simultaneously in single pituitary cells obtained from normal tissue. In addition to identifying somatotropes, the RHPA quantitates hormone secretion, which can be compared directly with intracellular calcium determinations. For dual-wavelength recordings, the ratio of the fluorescence intensities obtained with excitation a t 340 and a3t 80 nm wacsalculated This 340/380 ratio measures calcium in single cells essentially independent of dye content or cell thickness. After calcium recordings were completed, complement was added which develops lysis zones around thosecells that secreted GH
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