High-resolution differential interference contrast microscopy and digital imaging of the fluorescent calcium indicator dye fura-2 were performed simultaneously in single rat salivary gland acinar cells to examine the effects of muscarinic stimulation on cell volume and cytoplasmic calcium concentration ([Ca2+]i). Agonist stimulation of fluid secretion is initially associated with a rapid tenfold increase in [Ca2+]i as well as a substantial cell shrinkage. Subsequent changes of cell volume in the continued presence of agonist are tightly coupled to dynamic levels of [Ca2+]i, even during [Ca2+]i oscillations. Experiments with Ca2+ chelators and ionophores showed that physiological elevations of [Ca2+]i are necessary and sufficient to cause changes in cell volume. The relation between [Ca2+]i and cell volume suggests that the latter reflects the secretory state of the acinar cell. Agonist-induced changes in [Ca2+]i, by modulating specific ion permeabilities, result in solute movement into or out of the cell. The resultant cell volume changes may be important in modulating salivary secretion.

The localized increase cannot be detected by fura-2 but is sufficient to open the Ca2+-sensitive K+ channels located in the basolateral membrane. We concluded that a receptormobilized intracellular store of Ca2+ is localized at or near the basolateral membrane.Fluid secretion by mammalian exocrine cells, including salivary acinar cells, is regulated by alterations in plasma membrane ion permeabilities (1). Among the earliest events associated with agonist-induced stimulation offluid secretion are a rapid plasma membrane hyperpolarization (1-4) and a dramatic loss of cellular K+ (5). Patch-clamp studies have identified Ca2+-and voltage-sensitive K+ channels in salivary and pancreatic acinar basolateral membranes whose activation by secretagogues is believed to underlie these effects (6). Another early event associated with agonist stimulation is a rapid rise in the level of intracellular free calcium ([Ca2+]i) (7-11). As in a wide variety of other cell types, [Ca2+]i is thought to rise as a result of inositol trisphosphate mobilization of intracellular stores (9,12,13), as well as enhanced Ca2+ influx across the plasma membrane (7,(14)(15)(16)(17). A large body of evidence indicates that the rise of [Ca2+]J is directly responsible for activating the K+ channels in the acinar basolateral membranes. Specifically, it has been shown that (i) the Ca2+ sensitivity of the K+ channels is within the appropriate physiological (agonist-induced) range of [Ca2+]J (6, 18-21),(ii) the cellular K+ loss is abolished in Ca2+-depleted cells stimulated in Ca2+-free medium (22, 23), (iii) the application of Ca2" ionophores to acinar cells activates K+ channels and causes a loss of cellular K+ similar to that caused by agonists (23-26), (iv) the intracellular perfusion of a Ca2+ chelator blocks the agonist-stimulated increase in K+ conductance (18,26,27), and (v) the intracellular perfusion of inositol trisphosphate activates the K+ conductance (28).One aspect that has not yet been explored, however, is the temporal relationship between the agonist-induced rise of (29). In the present study, we examine the relationship between the agonist-induced rise of [Ca2+] (measured with the Ca2+-sensitive fluorescent dye fura-2) and membrane K+ conductance (measured simultaneously with an intracellular microelectrode) in rat parotid acinar cells. The data indicate that a receptor-mobilized store of Ca2+ is localized at or near the basolateral membrane.

Previously we reported the effects of postnatal castration on the postorganizational development of the sympathetic hypogastric ganglion (Hamill and Guernsey, 1983; Melvin and Hamill, 1986). "Postorganization" implies an activational role for gonadal hormones, in contrast to the permanent organizing effects that occur perinatally. We now report results that suggest that the major pelvic ganglion (PG), a mixed parasympathetic and sympathetic ganglion, is similarly regulated by testosterone during development. Choline acetyltransferase (CAT) and tyrosine hydroxylase (T-OH) activities were used to examine normal PG ontogeny. The normal development of these biochemical indices occurs primarily after day 10. Postnatal castration at 10-11 d of age completely prevented the postorganizational developmental increase of T-OH activity. At 12 postoperative weeks T-OH activity in castrates was approximately 6% that of the control animals (control, 2880 +/- 127 pmol/ganglion X hr; castrated, 161 +/- 16 pmol/ganglion X hr; p less than 0.001). In fact, by only 1 postoperative week, T-OH activity was already significantly reduced in castrated animals (control, 480 +/- 69 pmol/ganglion X hr; castrated, 179 +/- 6 pmol/ganglion X hr; p less than 0.001). CAT activity and total ganglion protein were also significantly reduced by 1 postoperative week. In contrast to T-OH activity, however, these indices continued to develop at diminished rates. By 12 postoperative weeks CAT activity and total ganglion protein in castrates were 30 and 50% of control values, respectively, resulting in a significant developmental abnormality in CAT-specific activity. Testosterone replacement reversed the castration-induced developmental deficits of T-OH and CAT activities.(ABSTRACT TRUNCATED AT 250 WORDS)

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