Timothy R. Cheek

Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiple second messenger systems.

A cloned seven transmembrane‐spanning Drosophila octopamine/tyramine receptor, permanently expressed in a Chinese hamster ovary cell line, both inhibits adenylate cyclase activity and leads to the elevation of intracellular Ca2+ levels by separate G‐protein‐coupled pathways. Agonists of this receptor (octopamine and tyramine), differing by only a single hydroxyl group in their side chain, may be capable of differentially coupling it to different second messenger systems. Thus, a single receptor may have a different pharmacological profile depending on which second messenger system is used to assay its efficacy.

Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2.

Temporal and spatial changes in the concentration of cytosolic free calcium ([Ca2+]i) in response to a variety of secretagogues have been examined in adrenal chromaffin cells using digital video imaging of fura‐2‐loaded cells. Depolarization of the cells with high K+ or challenge with nicotine resulted in a rapid and transient elevation of [Ca2+]i beneath the plasma membrane consistent with Ca2+ entry through channels. This was followed by a late phase in which [Ca2+]i rose within the cell interior. Agonists that act through mobilization of inositol phosphates produced an elevation in [Ca2+]i that was most marked in an internal region of the cell presumed to be the site of IP3‐sensitive stores. When the same cells were challenged with nicotine or high K+, to trigger Ca2+ entry through voltage‐dependent channels, the rise in [Ca2+]i was most prominent in the same localized region of the cells. These results suggest that Ca2+ entry through voltage‐dependent channels results in release of Ca2+ from internal stores and that the bulk of the measured rise in [Ca2+]i is not close to the exocytotic sites on the plasma membrane. Analysis of the time courses of changes in [Ca2+]i in response to bradykinin, angiotensin II and muscarinic agonists showed that these agonists produced highly heterogeneous responses in the cell population. This heterogeneity was most marked with muscarinic agonists which in some cells elicited oscillatory changes in [Ca2+]i. Such heterogeneous changes in [Ca2+]i were relatively ineffective in eliciting catecholamine secretion from chromaffin cells. A single large Ca2+ transient, with a component of the rise in [Ca2+]i occurring beneath the plasma membrane, may be the most potent signal for secretion.

Nicotine-evoked disassembly of cortical actin filaments in adrenal chromaffin cells

Using rhodamine‐phalloidin staining it was found that actin filaments are concentrated in the cortex of resting chromaffin cells. Cortical actin filaments were disassembled 15 s after stimulation by nicotine and had reassembled 30 s later. Actin filament disassembly following nicotinic stimulation was also detected using the DNase I inhibition assay. Disassembly was independent of external calcium, insensitive to trifluoperazine and was not elicited by high K+, muscarinic agonists or phorbol ester. Disassembly of cortical actin filaments may allow access of secretory granules to exocytotic sites and act in conjunction with a rise in intracellular free calcium to bring about the full secretory response due to nicotinic agonists.

Reorganisation of peripheral actin filaments as a prelude to exocytosis

Evidence is presented, from studies on the adrenal chromaffin cell, that reorganisation of the cortical actin network is necessary to allow granules to reach exocytotic sites in stimulated cells. This reorganisation may involve changes in actin filament cross-linking, assembly and interactions with secretory granule and plasma membranes. The possibility is discussed that cytoskeletal elements including the membrane-binding proteins caldesmon, p70 and p36 may be involved in granule-plasmalemmal interactions immediately prior to exocytosis.

Spatial localization of the stimulus-induced rise in cytosolic Ca2+ in bovine adrenal chromaffin cells: distinct nicotinic and muscarinic patterns

The spatial distribution of the intracellular free Ca2+ (Ca2+ i) rise elicited by different stimuli in bovine adrenal chromaffin cells was examined in single fura‐2‐loaded cells. In response to the potent secretagogues nicotine and high K+, Ca2+ i was initially localized exclusively to the entire subplasmalemmal area of the cell. In response to the ineffective secretagogues, methacholine and muscarine, the rise in Ca2+ i originated only in one pole of the cell and even at the peak of the response Ca2+ was still generally restricted to this same area of the cell. These results suggest that the triggering of exocytosis from these cells requires a specific spatial distribution of Ca2+ i.

Effect of activation of muscarinic receptors on intracellular free calcium and secretion in bovine adrenal chromaffin cells

Stimulation of the nicotinic receptor of bovine chromaffin cells results in a rise in intracellular free calcium [( Ca2+]i) and subsequent release of catecholamine. This response is totally dependent on the presence of external Ca2+. Monitoring [Ca2+]i using quin-2 demonstrated a rise in [Ca2+]i in response to muscarinic agonists which was approximately 4-times less than that obtained in response to nicotinic stimulation. This atropine-sensitive [Ca2+]i rise occurred after a 10-s lag and was found to be independent of the external Ca2+, implying the existence of an intracellular Ca2+ source. Despite producing this [Ca2+]i rise low concentrations of the muscarinic agonist, methacholine (under 1 X 10(-3) M), failed to trigger secretion itself and did not effect the secretory response elicited by nicotine. Challenging the cells with higher methacholine concentrations (over 1 X 10(-3) M) resulted in the same [Ca2+]i rise, no secretion, but an inhibition of secretion due to nicotine. This latter response, however, was found to be atropine-insensitive and therefore non-muscarinic. The [Ca2+]i rise and secretion due to depolarization by 55 mM K+ were largely unaffected by prior addition 1 X 10(-2) M methacholine, inferring that high concentrations of methacholine inhibit nicotine-induced secretion by interacting with the nicotinic receptor. These results provide evidence consistent with the existence of an intracellular Ca2+ store mobilized by muscarinic receptor activation in bovine chromaffin cells and show that, despite causing a rise in [Ca2+]i, bovine chromaffin cell muscarinic stimulation does not effect secretion itself or secretion induced by either nicotine or high K+.

Identification of a secretory granule-binding protein as caldesmon

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium1–3 which initiates catecholamine secretion by exocytosis4,5. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells6,7, and isolated granule membranes bind both calmodulin8 and a series of cytosolic proteins9–12 in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle13. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.

Chapter 26 Agonist-specific coupling of G-protein-coupled receptors to second-messenger systems

Publisher Summary This chapter presents evidences for the concept of agonist-specific coupling of G-protein-coupled receptors to multiple second-messenger systems. It discusses the related concept of synthetic ligand-specific coupling of G-protein-coupled receptors to second-messenger systems. The important general implications of these concepts for pharmacology and signalling mechanisms are examined in the chapter. Agonist-specific coupling of G-protein-coupled receptors to second-messenger systems may be a general property shared by a range of different receptors. A cloned type 1 pituitary adenylyl cyclase- activating polypeptide (PACAP) receptor and four spliced variant forms of the receptor have been reported to be differentially coupled to adenylate cyclase and phospholipase C by two naturally occurring forms of PACAP—namely, PACAP-27 and PACAP-38. Studies on several vertebrate cloned G-protein- coupled receptors have also demonstrated that a range of synthetic ligands that can act as agonists of these receptors may also be capable of coupling the receptors differentially to different second-messenger systems.

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration ([Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura‐2‐loaded cells and cell populations. In cell populations caffeine elicited a large (∼200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (> 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine‐sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine‐sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.

Calcium regulation and homeostasis.

The use of techniques to visualize the stimulus-induced changes in [Ca2+]i that occur at the single cell level has revealed that intracellular Ca2+ signals can be remarkably organized in space (waves), as well as in time (oscillations). New insights are beginning to emerge into how these complex Ca2+ signals may be generated, and into how Ca2+ signals may be transmitted from cell to cell.