James W. Putney

A model for receptor-regulated calcium entry

A model is proposed for the mechanism by which activation of surface membrane receptors causes sustained Ca2+ entry into cells from the extracellular space. Reassessment of previously published findings on the behavior of receptor-regulated intracellular Ca2+ pools leads to the conclusion that when such pools are empty, a pathway from the extracellular space to the pool is opened; conversely when the pool is filled, the pathway is closed and it becomes relatively stable to depletion by low Ca2+ media or chelating agents. The biphasic nature of agonist-activated Ca2+-mobilization is thus seen as an initial emptying of the intracellular Ca2+ pool by inositol (1,4,5) trisphosphate, followed by rapid entry of Ca2+ into the pool and, in the continued presence of inositol (1,4,5) trisphosphate, into the cytosol. On withdrawal of agonist, inositol (1,4,5) trisphosphate is then rapidly degraded, the pathway from the pool to the cytosol is closed, and rapid entry from the outside continues until the Ca2+ content of the pool reaches a level that inactivates Ca2+ entry. This capacitative model allows for Ca2+ release and Ca2+ entry to be controlled by a single messenger, inositol (1,4,5) trisphosphate.

Recent hypotheses regarding the phosphatidylinositol effect

Abstract In 1975, Michell first proposed that activation of phosphatidylinositol turnover provided a direct link between surface receptors and membrane Ca gates. Subsequently, a number of laboratories have begun to re-investigate this phenomenon first described by Hokin and Hokin some twenty years earlier. As would be expected, some new hypothesis have emerged, most being extensions or revisions of Michells original concept. Despite difficulties in obtaining direct proof, indirect evidence suggests that the plasma membrane is the primary locus of receptor-activated phosphatidylinositol turnover, at least for phosphatidylinositol breakdown and phosphatidic acid synthesis. The presence or absence of Na + can markedly affect labelling of phosphatidylinositol by radioactive precursors but there is no compelling evidence that the initial events are mediated by Na + . Prostaglandins are apparently formed in some tissues on receptor activation, but in most instances the evidence suggests that these compounds are not obligatory intermediates in tissues that show the phosphatidylinositol effect. Several laboratories have obtained evidence that phosphatidic acid newly synthesized following phosphatidylinositol breakdown, may function as an endogenous Ca ionophore under neurohumoral control.

A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland

Electrically permeabilized cells of rat parotid gland, prelabelled with [3H]-inositol, synthesized [3H]-inositol phosphates (IP3 and IP2) when stimulated with alpha 1-adrenergic, muscarinic-cholinergic, and substance P receptor-agonists. Non-hydrolyzable analogues of GTP (GTP gamma S and GppNHp) also stimulated [3H]-IP3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [3H]-IP3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca2+-mobilizing receptors to phospholipase C in parotid gland.

Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate.

Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.

Inositol lipids and cell stimulation in mammalian salivary gland

The rat parotid salivary gland shows marked alterations in phospholipid metabolism when stimulated by certain agonists. These agonists are those which cause cellular Ca mobilization by activation of muscarinic, alpha-adrenergic or peptidergic (substance P) receptors. The phospholipid changes apparently reflect the activation of a phosphoinositide-phosphatidic acid cycle, the precise pathways of which are not known with certainty. The observed effects include (1) an increased labelling by 32PO4 of phosphatidylinositol and phosphatidic acid, (2) net synthesis of phosphatidic acid, (3) net breakdown of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate. These effects apparently do not require the presence of extracellular Ca or the release of internal Ca and cannot be produced by the artificial introduction of Ca into the cytosol with Ca ionophores. These findings are consistent with the view that a receptor-mediated alteration in phosphoinositide metabolism represents an early step in the stimulus-response pathway in the parotid acinar cell. It has been suggested that phosphatidic acid synthesis might be of central importance in mediating Ca influx and that PIP2 breakdown might play a role in activation of Ca release. Evidence for these latter ideas is for the present largely circumstantial.

Activation by calcium of membrane channels for potassium in exocrine gland cells

In the rat parotid salivary gland, fluid secretion is regulated by alterations in fluxes of monovalent ions. In vitro, stimulation of muscarinic, alpha-adrenergic or substance P receptors provokes a biphasic increase in membrane permeability to K+ which can be conveniently assayed as efflux of 86Rb. The increased 86Rb flux is thought to arise in response to a receptor mediated elevation in [Ca2+]i which activates Ca2+-activated K+-channels. The biphasic nature of the response is presumably due to a biphasic mode of Ca2+ mobilization by secretagogues; a transient response reflects release of a finite pool of Ca from an intracellular store while a more sustained phase results from Ca entry through receptor operated Ca channels or gates. Calcium also mediates an increased Na+ entry which in turn activates the Na+, K+-pump. The mechanism involved in the regulation of monovalent ion channels by Ca2+ is not understood.

Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

Abstract The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (10−4M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate 86Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or 86Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by 10−5M indomethacin. Eicosatetraynoic acid (2×10−5M) inhibited 86Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (10−4M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands. In the exocrine glands activation by neurotransmitters (or analogs) of receptors that mobilize cellular Ca also stimulates the incorporation of 32PO4 into phosphatidylinositol (1–3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6–8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7–9). There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10–14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13, 14). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13, 14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16, 17). The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover — the rat parotid and lacrimal glands.