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Biomedical subjects

M J Berridge

Publications and source records attributed to M J Berridge.

At least 145 records · Page 8Linked to original sources
PubMed1983

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.

Animals↗
PubMed1983

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.

Animals↗
PubMed1982

Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.

1. The effect of Li+ on the agonist-dependent metabolism of [3H]inositol has been studied in rat brain, rat parotid and the insect salivary gland. 2. When brain or parotid slices were incubated in the presence of [3H]inositol, Li+ was found to amplify the ability of agonists such as carbachol, phenylephrine, histamine, 5-hydroxytryptamine and Substance P to elevate the amount of label appearing in the inositol phosphates. 3. A different approach was used with the insect salivary gland, which was prelabelled with [3H]inositol. After washing out the label, the subsequent release of [3H]inositol induced by 5-hydroxytryptamine was greatly decreased by Li+. During Li+ treatment there was a large accumulation of [3H]inositol 1-phosphate. 4. This ability of Li+ to greatly amplify the agonist-dependent accumulation of myo-inositol 1-phosphate offers a novel technique for identifying those receptors that function by hydrolysing phosphatidylinositol. 5. The therapeutic action of Li+ may be explained by this inhibition of myo-inositol 1-phosphatase, which lowers the level of myo-inositol and could lead to a decrease in the concentration of phosphatidylinositol, especially in those neurons that are being stimulated excessively. This alteration in phosphatidylinositol metabolism may serve to reset the sensitivity of those multifunctional receptors that generate second messengers such as Ca2+, cyclic GMP and the prostaglandins.

Animals↗
PubMed1982

Phosphatidylinositol-hydrolysing enzymes in blowfly salivary glands.

The salivary glands of adult blowflies (Calliphora erythrocephala) contain enzymes that hydrolyse phosphatidylinositol, predominantly by a Ca2+-independent deacylation, though a Ca2+-dependent phosphodiesterase (phospholipase C) activity could be detected. The deacylating enzymes could also hydrolyse phosphatidylcholine and phosphatidylethanolamine, and were secreted in the saliva. Homogenization of salivary glands prelabelled with [3H]inositol resulted in a rapid deacylation of the endogenous 3H-labelled phosphatidylinositol; this hydrolysis was unaffected by addition of 5-hydroxytryptamine to the homogenate.

Animals↗
PubMed1981

Electrophysiological evidence for the existence of separate receptor mechanisms mediating the action of 5-hydroxytryptamine.

The transepithelial potential recorded across the salivary gland of the blowfly Calliphora responded to 5-hydroxytryptamine (5-HT) with a complex depolarising response characterised by oscillatory activity at low doses. A rapid screening procedure for characterizing the electrophysiological response of the gland to 5-HT and its analogues was developed. It consisted of recording the potential during the gradual addition of the agent in the form of a ramp. 5-HT gave a characteristic electrophysiological signature which was then compared with that of other analogues. Tryptamine, bufotenine and dimethyltryptamine gave potential signatures very similar to those of 5-HT. Histamine, 4- and 5-fluoro-alpha-methyltryptamine gave s predominantly hyperpolarising response which is characteristic of the effect produced by cyclic AMP. Analogues with substituents at the 5-position (methyl, methoxy, chloro) and 4-hydroxytryptamine gave a depolarising response indicative of a predominantly calcium effect. It is concluded that 5-HT acts through separate transducing mechanisms. The depolarizing response seems to indicate an action through calcium whereas the hyperpolarizing response might be mediated through cyclic AMP. Whether or not these separate responses are linked to different 5-HT receptors remains to be determined.

Animals↗
PubMed1981

Separate 5-hydroxytryptamine receptors on the salivary gland of the blowfly are linked to the generation of either cyclic adenosine 3',5'-monophosphate or calcium signals.

1 5'-Hydroxytryptamine (5-HT) stimulates the formation of two separate second messengers in the salivary gland of the blowfly. Activation of adenylate cyclase raises adenosine 3',5'-monophosphate (cyclic AMP) whereas the hydrolysis of phosphatidylinositol (PI) is associated with an increase in calcium permeability. The possibility that these two signal pathways might be controlled by separate 5-HT receptors was studied by testing the specificity of 5-HT analogues and antagonists. 2 The parent compound 5-HT was found to stimulate both cyclic AMP formation and the related parameters of PI hydrolysis and calcium transport with similar dose-response relationships. 3 Certain analogues such as 4- and 5-fluoro-alpha-methyltryptamine were capable of raising cyclic AMP levels and stimulating fluid secretion but did not stimulate the hydrolysis of PI or the entry of calcium. 4 Other analogues, which had chloro or methyl substituents at the 5-position, were found to stimulate the hydrolysis of PI and the transport of calcium at much lower doses than those required to stimulate the formation of cyclic AMP. 5 Antagonists were also found to exert selective effects. Methysergide was a potent inhibitor of PI hydrolysis whereas cinanserin was far more selective in blocking the stimulatory effect of 5-HT on cyclic AMP formation. 6 It is concluded that 5-HT acts on two separate receptors, a 5-HT1 receptor acting through calcium and a 5-HT2 receptor which mediates its effects through cyclic AMP.

Animals↗
PubMed1981

Phosphatidylinositol hydrolysis and calcium signaling.

The salivary glands of the blowfly possess two separate 5-HT receptors. One receptor system is linked to adenylate cyclase to produce cyclic AMP, which regulates cation transport across the epithelium. The other receptor acts through calcium, which stimulates anion transport. Activation of this second receptor is associated with the specific hydrolysis of phosphatidylinositol (PI) to give a PI response that appears to be a characteristic feature of receptors that act through calcium. Studies on this insect gland have established a close correlation between PI hydrolysis and changes in calcium gating. The idea that these two receptor-mediated events might be intimately related was strengthened by the observation that the ability of 5-HT to gate calcium could be varied by adjusting the level of PI in the membrane.

Animals↗
PubMed1980

Changes in cyclic AMP and cyclic GMP concentrations during the action of 5-hydroxytryptamine on an insect salivary gland.

Salivary glands from adult blowflies (Calliphora erythrocephala Meigen) were studied in vitro. The time course of changes in cyclic AMP content of the glands was followed at different concentration of 5-hydroxytryptamine. There was an immediate biphasic rise and fall in cyclic AMP content, following by a slower rise and subsequent gradual decline. The initial rise preceded the onset of fluid secretion by the glands. Rises in cyclic AMP content were inhibited by compound RMI 12330 A (an adenylate cyclase inhibitor) and were halted after about 15-20s if the glands were deprived of Ca2+. Theophylline (a phosphodiesterase inhibitor) abolished the decline phase of the fast response, Losses of cyclic AMP from the glands either to the bathing medium or to the saliva were small and could not account for the rapid fall found. Evidence is presented that cyclic GMP is not involved in the process of initiating secretion in the blowfly salivary gland.

Adenylyl Cyclase Inhibitors↗
PubMed1979

Relationship between phosphatidylinositol synthesis and recovery of 5-hydroxytryptamine-responsive Ca2+ flux in blowfly salivary glands.

Each salivary gland contains about 135 pmol of phosphatidylinositol. In glands prelabelled by incubation for 1 h with [32P]Pi or [3H]inositol there was a subsequent breakdown of 80% of the labelled phosphatidylinositol over a 2 h incubation period with 10 micrometer-5-hydroxytryptamine. However, there was no detectable decrease either in total phosphatidylinositol based on phosphorus analysis by chemical estimation or in the radioactivity of [32P]phosphatidylinositol in salivary glands of flies raised from the larval stage on diets containing[32P]Pi and whose phospholipids were uniformly labelled. These results suggest that the pool of phosphatidylinositol involved with Ca2+ gating is a small fraction of the total phosphatidylinositol content. Furthermore it is this small compartment that is preferentially radioactively labelled during short-term incubations with radioactively labelled precursors. In salivary glands incubated for 2 h with 10 micrometer-5-hydroxytryptamine there was a marked decrease in the flux of 45Ca2+ across the gland. After removal of the hormone, incubation of salivary glands for 1 h in the presence of 2mM-inositol, but not choline or ethanolamine, resulted in a recovery of hormone-responsive 45Ca2+ flux. Quantitative studies revealed that less than 9 pmol of phosphatidylinositol must be formed to fully restoret he 5-hydroxytryptamine-responsive 45Ca2+ flux.

Biological Transport↗
PubMed1979

Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland.

The addition of 5-hydroxytryptamine to the isolated blowfly salivary gland stimulates fluid secretion, transepithelial calcium transport and the breakdown of 32P- or 3H-labelled phosphatidylinositol The breakdown of [32P]phosphatidylcholine and [32P]-phosphatidylethanolamine was not stimulated by 5-hydroxytryptamine. In salivary glands incubated with myo-[2-3H]inositol for 1--3 h, more than 95% of the label retained by the tissue was in the form of phosphatidylinositol. The addition of 5-hydroxytryptamine resulted in an increase in the accumulation of label in intracellular inositol 1:2-cyclic phosphate, inositol 1-phosphate and free inositol along with an increase in the release of [3H]inositol to the medium and saliva. The release of [3H]inositol to the medium served as a sensitive indicator of phosphatidylinositol breakdown. The release of [3H]inositol was not increased by cyclic AMP or the bivalent-cation ionophore A23187 under conditions in which salivary secretion was accelerated. The stimulation of fluid secretion by low concentrations of 5-hydroxytryptamine was potentiated by 3-isobutyl-1-methylxanthine, which had no effect on inositol release. The stimulation of fluid secretion by 5-hydroxytryptamine was greatly reduced in calcium-free buffer, but the breakdown of phosphatidylinositol continued at the same rate in the absence of calcium. These results support the hypothesis that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium.

Animals↗
PubMed1979

Inhibition of phosphatidylinositol synthesis and the inactivation of calcium entry after prolonged exposure of the blowfly salivary gland to 5-hydroxytryptamine.

The incorporation of [32P]Pi into all salivary-gland phospholipids except phosphatidic acid was inhibited by 5-hydroxytryptamine. The accumulation of [32P]Pi into phosphatidic acid was actually enhanced by 5-hydroxytryptamine. There was an inhibition of labelled inositol incorporation into phosphatidylinositol by 5-hydroxytryptamine, which seems to be mediated by calcium because it was mimicked by the ionophore A23187, but was prevented if glands were stimulated with 5-hydroxytryptamine in the absence of external calcium. Inhibition of synthesis together with stimulation of breakdown will decrease the concentration of phosphatidylinositol, which could account for the inactivation of calcium transport observed at high 5-hydroxytryptamine concentrations. When salivary glands were stimulated with 1 micrometer-5-hydroxytryptamine, there was a rapid increase in the transfer of 45Ca2+ from the medium into the saliva, but with time this transport declined to a low value. If the glands were washed free of 5-hydroxytryptamine and incubated in the presence of 2mM-inositol for 1 h, the increase in calcium transport caused by 5-hydroxytryptamine was restored. There was little recovery in the absence of inositol. If glands were stimulated with 5-hydroxytryptamine in the absence of external calcium, a condition which prevents the inhibition of phosphatidylinositol synthesis, calcium transport in response to 5-hydroxytryptamine was greater than in glands preincubated with 5-hydroxytryptamine in the presence of calcium. The inactivation of calcium transport may result from a decrease in phosphatidylinositol concentration. These results support the hypothesis that the hydrolysis of phosphatidylinositol plays some role in either the opening or closing of calcium 'gates'.

Animals↗
PubMed1979

A comparative survey of the function, mechanism and control of cellular oscillators.

This review attempts to survey in a uniform manner the available evidence concerning the generation and behaviour of several well-investigated cellular oscillators. Members of two broad classifications are contrasted: (i) cytoplasmic oscillations, where the periodic phenomena is generated by an instability pathway and (ii) membrane oscillators in which a membrane potential rhythm is generated at the membrane. Interactions between the cytoplasmic and membrane compartments are considered and the effects of these interactions on oscillatory behaviour is discussed. Because of their biological importance and the greater body of experimental results, particular attention is directed to a study of membrane potential oscillations. These systems can be approximately classified in two groups: (i) systems in which a periodic potential results from oscillatory changes in permeability and (ii) systems in which potential oscillations result from the periodic activity of an electrogenic pump. The examples considered include the glycolytic oscillator, oscillations in vein contraction in the slime mould Physarum polycephalum, rhythmic aggregation in Dictyostelium discoideum, neural oscillators, the periodic potential in Purkinje fibres and the sino-atrial node and rhythmic behaviour in smooth muscle. Questions considered include the generation of periodic activity, the modulation of the oscillation by drugs and other metabolic and membrane effectors and the question of the functional role of these oscillations.

Adrenal Cortex↗