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Biomedical subjects

J W Putney

Publications and source records attributed to J W Putney.

At least 145 records · Page 8Linked to original sources
PubMed1982

Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells.

The metabolism of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in rat parotid acinar cells was investigated, particularly with regard to the effects of receptor-active agonists. Stimulation of cholinergic-muscarinic receptors with methacholine provoked a rapid disappearance of 40--50% of [32P]PtdIns(4,5)P2, but had no effect on PtdIns4P. Adrenaline, acting on alpha-adrenoceptors, and Substance P also stimulated net loss of PtdIns(4,5)P2. The beta-adrenoceptor agonist, isoprenaline, and the Ca2+ ionophore, ionomycin, failed to affect labelled PtdIns(4,5)P2 or PtdIns4P. By chelation of extracellular Ca2+ with excess EGTA, and by an experimental protocol that eliminates cellular Ca2+ release, it was demonstrated that the agonist-induced decrease in PtdIns(4,5)P2 is independent of both Ca2+ influx and Ca2+ release. These results may suggest that net PtdIns(4,5)P2 breakdown is an early event in the stimulus-response pathway of the parotid acinar cell and could be directly involved in the mechanism of agonist-induced Ca2+ release from the plasma membrane.

Animals↗
PubMed1982

Regulation of phosphatidate synthesis by secretagogues in parotid acinar cells.

The metabolism of phosphatidate in rat parotid acinar cells was investigated, particularly with regard to the actions of agonists known to act by mobilizing Ca2+. When cells were incubated in medium containing 10 microM-[32P]Pi, phosphatidate was rapidly labelled, approaching an apparent steady-state with a half-time of approx. 20 min. Methacholine provoked a more than doubling of phosphatidate radioactivity, which was reversed by the muscarinic antagonist atropine. These results suggest that phosphatidate labels to near steady-state rapidly and that in cells prelabelled for 60 min the increase in radioactivity induced by agonists probably reflects net synthesis rather than an increase in specific radioactivity. Phosphatidate synthesis in response to methacholine was rapid and occurred, within the resolution of a few seconds, with no measurable latency. Adrenaline and substance P also stimulated phosphatidate synthesis but both agonists were less efficacious than methacholine. A Ca2+ ionophore, ionomycin, did not provoke phosphatidate synthesis. By using a protocol that eliminates the receptor-regulated Ca2+ pool, it was demonstrated that methacholine-induced phosphatidate formation does not come about as a consequence of Ca2+ influx nor of Ca2+ release. These results indicate that the phosphatidate synthesis response has characteristics compatible with its previously suggested role as a primary mediator of membrane Ca2+-gating.

Animals↗
PubMed1982

Net calcium fluxes in rat parotid acinar cells: evidence for a hormone-sensitive calcium pool in or near the plasma membrane.

The effects of receptor stimulation on net fluxes of 45Ca in parotid acinar cells were investigated. When cellular 45Ca content was near steady-state, muscarinic receptor activation by carbachol caused a net efflux of 45Ca (not always reproducible) followed by a net influx. In the presence of excess ethylene glycol bis (beta-aminoethyl ether) N,N,N,N-tetraacetic acid, net efflux invariably occurred, but the influx phase was prevented. When the muscarinic receptor antagonist atropine was added to the medium during the influx phase, an abrupt transient influx occurred followed by a return of net influx to the prestimulation level. When cellular responses believed to reflect intracellular ionized Ca (k + permeability, protein secretion) were examined under similar conditions, atropine invariably had an inhibitory effect. The Ca taken up in response to atropine apparently replenishes the hormone-sensitive pool of cellular Ca since it can be released subsequently by adrenoceptor activation. Taken together, these observations suggest that when atropine is administered to cholinergically activated cells, the hormone-sensitive Ca pool rapidly refills from the extracellular fluid without a concomitant increase in ionized intracellular Ca. Thus, it is suggested that this Ca pool is most likely associated with the plasma membrane.

Animals↗
PubMed1982

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.

Animals↗
PubMed1982

Nature of the receptor-regulated calcium pool in the rat parotid gland.

1. The transient release of 86Rb from parotid slices induced by secretagogues was investigated. 2. In the absence of external Ca, only one transient response (to carbachol) could be obtained. 3. After blocking the cholinergic stimulus with atropine, a second response (to substance P) could be elicited if the slices were briefly (2 min) exposed to a Ca-containing medium. 4. The magnitude of the substance P response depended on the concentration of Ca to which the slices had been exposed. 5. An exposure to Ca of 2 min duration was found to be sufficient to restore maximal substance P responsiveness. 6. These results are interpreted to mean that the cholinergic stimulus elicited a transient 86Rb efflux response by first releasing a finite pool of cellular Ca which could be reloaded from the extracellular space by a brief (2 min) incubation in a Ca-containing medium. The magnitude of the subsequent response to substance P apparently reflects the quantity of Ca taken up by the pool. 7. A number of cationic substances antagonized the restoration by Ca of the substance P response; the rank order of potency was: La3+ = Tm3+ greater than Co2+ = Ni2+ greater than neomycin much greater than Mg2+. 8. These same substances were examined for their relative abilities to inhibit Ca binding to phosphatidylinositol-4, 5-bisphosphate; in this case the rank order of potency was: La3+ = Tm3+ greater than neomycin greater than Co2+ greater than Ni2+ = Mg2+. 9. It is concluded that the uptake process does not appear to reflect Ca binding to phosphatidylinositol-4, 5-bisphosphate.

Animals↗
PubMed1982

Actions of ionomycin in rat parotid gland.

The effects of ionomycin (SQ 23,377), a carboxylic acid Ca-ionophore, on the rat parotid acinar cell were investigated. Ionomycin stimulated 86Rb efflux from parotid slices and was substantially more potent and efficacious than the Ca-ionophore, A-23187. The release of 86Rb was dependent on the concentration of ionomycin and of Ca. Ionomycin also stimulated 22Na uptake and 3H-protein secretion, but did not stimulate the incorporation of 32PO4 into phosphatidylinositol. These observations are consistent with an action of ionomycin in increasing cytosolic Ca by acting as an ionophore and not involving endogenous receptors. Pretreatment with ionomycin inhibited the transient, Ca-independent responses to carbachol or physalaemin. When ionomycin was added to parotid cells pre-equilibrated with 45Ca, a net loss of radiocalcium was observed. These observations suggest that ionomycin can release the receptor-regulated cellular Ca pool. Morphological studies did not reveal any nonspecific deleterious effects in the cells after incubation with 2.67 microM ionomycin.

Animals↗
PubMed1981

Receptor regulation of calcium release and calcium permeability in parotid gland cells.

The mechanism by which hormones and neurotransmitters regulate fluid secretion in exocrine glands apparently involves the regulation of transmembrane movements of electrolytes, a process for which Ca serves as a second messenger. Analysis of the kinetics of efflux of 86Rb+ (a marker for K+) indicates that the initial phase of the response to secretagogues is mediated through the release of Ca from a cellular pool inaccessible to chelating agents. By investigating the movements of 45Ca under nearly steady-state conditions, we find that this cellular pool can be filled from the extracellular space without a concomitant elevation in ionized intracellular Ca2+. This suggests that the cellular pool is probably associated with the plasma membrane. We have also investigated the possible role of phosphatidic acid in the mechanism by which receptors mobilize Ca2+. Our results suggest that phosphatidic acid, formed on receptor activation, may directly mediate Ca influx into the acinar cell.

Animals↗
PubMed1981

The relationship of phosphatidylinositol turnover to receptors and calcium-ion channels in rat parotid acinar cells.

To help elucidate the possible role of phosphatidylinositol in the regulation of membrane permeability to Ca2+, the relationship in the rat parotid gland of phosphatidylinositol turnover to hormone receptor binding and to the hormone-mediated increase in K+ permeability (a Ca2+-dependent phenomenon) was investigated. The concentrations of adrenaline and substance P required to stimulate phosphatidylinositol turnover were found to be similar to those required for the Ca2+-mediated change in K+ permeability and for ligand binding. However, in the case of muscarinic (cholinergic) receptor stimulation, the phosphatidylinositol response was better correlated to the increase in membrane permeability to Ca2+, as determined by the change in K+ permeability, than to receptor occupation. Consistent with this relationship between the phosphatidylinositol response and Ca2+-channel activation were results obtained by simultaneous administration of maximal or submaximal concentrations of muscarinic and alpha-adrenergic agonists. The extent of 32P incorporation when stimulated by maximal concentrations of two agonists did not summate, but, rather, was intermediate between the response of either agonist alone. One interpretation for these observations is that the phosphatidylinositol response may not be related to receptor occupation or activation, but may be involved in the Ca2+-gating mechanism itself.

Animals↗
PubMed1980

Effect of carbachol on radiosodium uptake by dispersed pancreatic acinar cells.

The effects of carbachol on uptake of 22Na by enzymatically dispersed rat pancreatic acinar cells were determined. Carbachol caused a slight but significant increase in uptake of 22Na by the cells in the presence of absence of ouabain (10(-3) M). A maximal response was obtained with 10(-6) M carbachol. The effects of carbachol were blocked by 10(-5) M atropine. Caerulein (10(-7) M) also stimulated 22Na uptake, while epinephrine (10(-4) M) and substance P (10(-7) M) did not. Carbachol did not stimulate 22Na uptake in the absence of extracellular Ca, although Ca omission significantly elevated basal 22Na uptake. The divalent cationophore A-23187 caused Ca-dependent 22Na uptake at 20 microM concentration but not at 0.3 microM. These results, when considered with earlier reports by others, suggest that muscarinic receptor activation leads to an increase in permeability of the acinar cell membrane to Na, and that Ca may be second messenger for this effect.

Absorption↗
PubMed1980

The relationship between muscarinic receptor binding and ion movements in rat parotid cells.

1. The relationship of muscarinic receptor binding to ion fluxes induced by muscarinic agonists was investigated in rat parotid cells. 2. Receptor binding was measured with [3H]quinuclidinyl benzilate in living, dispersed parotid acinar cells. These same cells were used to determine concentration-effect relationships (ion fluxes) for agonists. 3. Receptor binding of antagonists accurately reflected the pharmacologically determined affinities. In the case of agonists, apparent pharmacological affinities were greater than binding affinities by a factor of 12.5 for methacholine and 18.7 for carbachol. 4. The reason for the discrepancy between agonist binding and effect is not known with certainty, but the existence of "spare" receptors is considered a possibility. 5. The total number of muscarinic receptors is estimated to be 23,000/cell. If some are spare, as few as 1800/cell may suffice to fully activate the available Ca channels.

Animals↗
PubMed1980

Binding of 125I-physalaemin to rat parotid acinar cells.

1. The binding of [125I]physalaemin to rat parotid acinar cells was investigated. 2. The [125I]physalaemin exhibited a small degree of specific binding that was rapid, reversible and saturable. 3. The EC50 values for inhibition of binding by four peptides were well correlated with their ability to activate Ca-dependent K release from the rat parotid gland. 4. The number of binding sites, which may represent substance P receptors, was estimated to be in the range of 200/cell, a value quite different from those reported previously for muscarinin (840/cell) or alpha-adrenergic (15,000/cell) receptors. 5. It is concluded that if, as previously suggested, these receptors regulate the same population of Ca channels, then the mechanism or perhaps efficiency by which this is achieved may differ for the three receptors.

Animals↗
PubMed1980

Stimulus-permeability coupling in rat lacrimal gland.

Incubation of isolated cells wtih 10(-2) M ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid or cobalt inhibits 22Na and 45Ca uptake stimulated by carbachol. The artificial introduction of Ca into the cytosol by the cation ionophore A23187 also initiates the 22Na uptake. Amiloride (10(-5) M) partially inhibits 22Na uptake induced by carbachol, but has no effect on receptor-stimulated 45Ca uptake or 86Rb release. Tetrodotoxin (TTX) has no effect on 22Na uptake stimulated by carbachol, whereas methoxyverapamil (D 600) produces a small but significant decrease in both 22Na and 45Ca uptake. This effect of D 600 may be related to a block of receptor activation and not to a block of Ca channel activation. Incubation in high K (56 mM) does not prevent the change in membrane permeability to Ca, K, and Na initiated by carbachol. It is concluded that carbachol stimulates the influx of Ca; the rise in the cytosolic Ca concentration then couples receptor activation to a change in membrane permeability to K and Na. The permeability mechanisms for Ca, K, and Na that are activated by carbachol appear to be specific for each of the three cations and appear to be dissimilar to permeability mechanisms in excitable tissue that carry the same ions.

Animals↗
PubMed1980

Muscarinic and alpha-adrenergic stimulation of Na and Ca uptake by dispersed lacrimal cells.

Rat lacrimal gland acinar cells were isolated and observed to be physiologically stable for several hours of incubation in vitro. With a double-isotope technique, it was found that carbachol and epinephrine stimulated the uptake of 22Na and 45Ca by lacrimal cells. These respnses were maximal at agonist concentrations of 10(-5) M and were blocked by atropine and phentolamine, respectively. It is concluded that muscarinic and alpha-adrenergic receptor activation increase the membrane permeability of the lacrimal gland acinar cell to Na and Ca, ions that may be important in the secretion of water by the lacrimal gland.

Absorption↗