Long Distance Communication between Muscarinic Receptors and Ca2+ Release Channels Revealed by Carbachol Uncaging in Cell-attached Patch Pipette

Abstract

We have investigated the characteristics of cytosolic Ca 2؉ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca 2؉ waves and local apical Ca 2؉ spikes. This is the first evidence for local Ca 2؉ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca 2؉ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca 2؉ spikes in the apical pole more than 15 m away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca 2؉ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca 2؉ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca 2؉ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca 2؉ release channels.In response to agonist stimulation, many cell types produce transient elevations in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ), which remain localized to a specific subcellular domain (1). In isolated pancreatic acinar cells, a low concentration of acetylcholine (ACh) 1 can evoke repetitive local cytosolic Ca 2ϩ spikes, which are confined to the granule containing apical pole (2). These local Ca 2ϩ spikes can activate exocytosis through the apical plasma membrane (3) and open Ca 2ϩ -dependent Cl Ϫ channels present exclusively in the apical plasma membrane, thereby regulating acinar fluid secretion (4).Agonist-receptor interaction causes activation of phospholipase C and the generation of inositol 1,4,5-trisphosphate (IP 3 ), which in turn opens Ca 2ϩ release channels in the endoplasmic reticulum membrane (5), thereby explaining the liberation of Ca 2ϩ from this intracellular store (6). Because the G-protein coupled receptors for cholecystokinin are localized in the basolateral plasma membrane as demonstrated in early autoradiographic studies on intact pancreas (7), the classical view of pancreatic acinar cell stimulation involves the binding of agonist to plasma membrane receptors located at the base. It is this end of the cell that is close to blood vessels and pancreatic nerve terminals from where physiologically released agonists approach the cell (8). If agonist-receptor interaction occurs at the base and Ca 2ϩ release occurs at the opposite (apical) pole, there is a need for a long distance Ca 2ϩ -releasing intracellular messenger. The established Ca 2ϩ -releasing messenger IP 3 (9, 10) as well as the more recently discovered Ca 2ϩ -liberating agents cyclic ADP-ribose and nicotinic acid adeni...

NAADP, cADPR and IP3 all release Ca2+ from the endoplasmic reticulum and an acidic store in the secretory granule area

“…Two NAADP-sensitive Ca 2+ stores in pancreatic acinar cells receptors, specifically and exclusively located in a small region at the base, induces initiation of cytosolic Ca 2+ signals in the apical part of the cell (Ashby et al, 2003a). Ca 2+ -induced Ca 2+ release can only be initiated in the secretory granule area .…”

ARC Channels: A Novel Pathway for Receptor-Activated Calcium Entry

“…For example, in polarized epithelial cells (such as parotid and pancreatic acinar cells), oscillatory [Ca 2+ ] i signals are initiated at specific "trigger zones" located in the apical region of the cell. Significantly, this phenomenon can be readily activated by local stimulation of appropriate receptors at the basal end of the cell (1). For these signals to be influenced by Ca 2+ entry via the ARC channels, either Ca 2+ would have to diffuse from basally activated ARC channels to the apical trigger zone or arachidonic acid would have to diffuse from its site of generation, presumably close to the basal receptors, to activate apically localized ARC channels.…”

Inositol 1,4,5-trisphosphate receptors (InsP3R)

“…The classical pathway linking stimulation by neurotransmitters and hormones to changes in phosphoinositide metabolism and the subsequent InsP 3 induced Ca 2+ release was in large part documented by work carried out in exocrine cells, in particular, acinar cells isolated from the exocrine pancreas (49). In seminal work using permeabilized rat pancreatic acini, Streb and colleagues demonstrated that the addition of InsP 3 resulted in Ca 2+ release from a nonmitochondrial Ca 2+ store (43).…”

“…The fidelity and specificity of the Ca 2+ signal required for exocytosis is thought to be largely determined by the differential expression, localization and modulation exhibited by the 3 InsP 3 R isoforms (14). For the most part, InsP 3 Rs are predominantly localized to the ER, although the golgi, nucleus, plasma membrane, peroxisomes and endolysosomal vesicles have also been reported to express small levels of InsP 3 Rs. Pancreatic acinar cells are highly polarized, both functionally and morphologically and InsP 3 Rs are predominantly expressed in ER extensions in the apical regions juxtaposed to the acinar lumen (22,24,32,57 …”

“…The target protein for InsP 3 binding was later identified as the inositol 1,4,5-trisphosphate receptor (InsP 3 R) (45). Strikingly, the secretion of digestive enzymes from pancreatic acinar cells has been shown to be entirely dependent on the activation of InsP 3 Rs and the resulting elevation in intracellular [Ca 2+ ]…”