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At least 757 records · Page 42Linked to original sources
PubMed1982

Myosin phosphorylation, agonist concentration and contraction of tracheal smooth muscle.

Myosin phosphorylation plays an important part in excitation--contraction coupling in smooth muscle. Phosphorylation by a Ca2+, calmodulin-dependent kinase stimulates the actin-activated Mg2+-ATPase activity of smooth muscle myosin, suggesting that myosin phosphorylation regulates smooth muscle contraction. This hypothesis is supported by evidence that myosin is phosphorylated during contraction and dephosphorylated during relaxation of intact smooth muscles stimulated with a single agonist concentration. However, there is little information regarding the response to stimulation with various agonist concentrations. As the dose-response relationships for phosphorylation and tension should be similar if myosin phosphorylation does, in fact, regulate smooth muscle contraction, we studied myosin phosphorylation in tracheal smooth muscle stimulated with a broad range of concentrations of the cholinergic agonist, methacholine. The results of these experiments are consistent with the hypothesis that myosin phosphorylation regulates smooth muscle contraction but they indicate a relatively complex relationship between myosin phosphorylation and the generation of isometric tension.

Adenosine Triphosphatases↗
PubMed1989

Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells.

Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.

Adrenal Medulla↗
PubMed1988

Dose-response of inhaled diltiazem on airway reactivity to methacholine and exercise in subjects with mild asthma.

Methacholine challenges were performed by 10 asthmatic subjects, 2 hours before and 15 minutes after placebo (diluent alone) and 5, 10, 15, 30, and 60 mg inhaled diltiazem given in a single-blind crossover manner. There was no significant change from placebo in the dose of methacholine required to produce a 20% decrease in forced expiratory volume in the first second (FEV1) (PD20); the fold increase in PD20 from baseline was 1.1 +/- 0.1 after placebo, 1.4 +/- 0.2 after 5 mg, 1.8 +/- 0.3 after 10 mg, 1.4 +/- 0.2 after 15 mg, 1.6 +/- 0.2 after 30 mg, and 1.2 +/- 0.1 after 60 mg. There was a 1% chance that we missed a twofold difference between placebo and the 10 mg dose because of inadequate sample size. Fifteen minutes before a standardized exercise challenge, 10 subjects received placebo, 10 mg, and the highest dose tolerated during the methacholine study (20 to 45 mg) in a randomized double-blind crossover design. The mean +/- SE maximum postexercise decrease in FEV1 was 28.8% +/- 5.7% after placebo, 23.4% +/- 4.6% after 10 mg, and 20.8% +/- 3.0% after high-dose diltiazem (P greater than 0.05). There was a 12% chance that we missed a 15% difference between placebo and the high-dose regimen because of inadequate sample size. We conclude that diltiazem does not attenuate airway reactivity to methacholine or exercise even when high concentrations are delivered to the lungs.

Adult↗
PubMed1988

Ocular autonomic nerve function in proliferative diabetic retinopathy.

Ocular autonomic nerve function was assessed in 28 diabetic patients with proliferative retinopathy (PDR) and 61 age- and sex-matched control subjects, by measurement of the pupil cycle time and determination of autonomic denervation hypersensitivity of the iris. Sustained pupil cycle time was absent in 88.5 per cent of PDR compared with 9.8 per cent of control subjects (p less than 0.001). Pupil constriction in response to 2.5 per cent methacholine--indicative of parasympathetic denervation hypersensitivity--was significantly increased in PDR (p less than 0.001), whilst pupil dilation in response to 0.5 per cent phenylephrine--indicative of sympathetic denervation hypersensitivity--was also significantly higher in PDR (p less than 0.001). Pupil reflexes were abnormal in 88.5 per cent of diabetics with proliferative retinopathy, with established autonomic denervation hypersensitivity in 57 per cent of patients.

Adult↗
PubMed1989

Autonomic denervation hypersensitivity in the primary glaucomas.

Autonomic denervation hypersensitivity of the iris was assessed in 44 patients with primary closed-angle glaucoma, 20 patients with primary open-angle glaucoma, and 40 age- and sex-matched control subjects. Significantly increased pupillary constriction in response to 2.5% methacholine chloride, indicating parasympathetic denervation hypersensitivity, was present in both closed-angle (p less than 0.02) and open-angle glaucoma patients (p less than 0.001), compared with the control group. Significantly increased pupillary dilatation in response to 0.5 per cent phenylephrine hydrochloride, indicating sympathetic denervation hypersensitivity of the iris, was also present in both closed-angle glaucoma (p less than 0.01) and open-angle glaucoma patients (p less than 0.05). The association between autonomic neuropathy and the primary glaucomas is discussed, with particular reference to the potential effects of autonomic dysfunction on aqueous dynamics.

Aged↗
PubMed1973

Biosynthesis of respiratory-tract mucins. A comparison of canine epithelial goblet-cell and submucosal-gland secretions.

1. A modified canine tracheal organ culture system was used to investigate differences between mucous secretions of epithelial goblet cells and the submucosal glands. 2. Denuded explants were prepared by removing goblet, ciliated and basal cells from the surface epithelium leaving an intact basement membrane and viable submucosa. 3. Denuded explants actively incorporated radioactive precursors into secreted macromolecules when cultured in medium 199 containing label. 4. Chromatography on Bio-Gel A-150m and electrophoresis on 1% agarose gels indicated that epithelial goblet cell secretions were relatively more sulphated than submucosal glandular secretions. 5. The glandular structures were shown to respond to a parasympathomimetic agent.

Animals↗
PubMed1974

Breakdown of phosphatidylinositol provoked by muscarinic cholinergic stimulation of rat parotid-gland fragments.

When rat parotid fragments that had been labelled with (32)P in vivo were exposed to high concentrations of acetylcholine, radioactivity was lost from phosphatidylinositol but not from other phospholipids. Simultaneously the concentration of phosphatidylinositol in the tissue decreased. If previously unlabelled tissue was incubated with (32)P(i) an increase in incorporation of radioactivity into phosphatidylinositol was observed during this decrease in concentration. The effects of acetylcholine were blocked by atropine, but not by tubocurarine. The response to acetylcholine was rapid, with up to one-third of the tissue's phosphatidylinositol disappearing within 5min. Similar effects were evoked by stimulation with methacholine and by high concentrations of tetramethylammonium ion; these responses were also atropine-sensitive and tubocurarine-insensitive. It is concluded that the event in inositol lipid metabolism that is affected by acetylcholine stimulation is removal of the phosphorylinositol group from the molecule; this is mediated through muscarinic cholinergic receptors. This is followed by a compensatory increase in the rate of synthesis of phosphatidylinositol, which has been described in detail in the past. These observations are compared with those of previous workers and are discussed in relation to the existing hypotheses relating to the significance of stimulus-provoked phosphatidylinositol turnover.

Acetylcholine↗
PubMed1976

Muscarinic cholinergic stimulation of phosphatidylinositol turnover in the longitudinal smooth muscle of guinea-pig ileum.

1. The metabolism of phosphatidylinositol and phosphatidate was investigated in fragments of longitudinal smooth muscle from guinea-pig ileum incubated with cholinergic and anticholinergic drugs. 2. Incorporation of Pi into these lipids was enhanced by acetylcholine and carbamoylcholine. 3. The receptor responsible for triggering this response was of the muscarinic type, since (a) the response was also produced by the muscarinic agonists acetyl-beta-methylcholine, carbamoyl-beta-methylcholine and pilocarpine, and (b) the response was prevented by atropine and prophylbenzilylcholine mustard, but not by tubocurarine. 4. Increased phosphatidylinositol labellin was clearly observed within 5 min in tissue treated with a high concentration of carbamoylcholine. 5. Halfmaximal stimulation of phosphatidylinositol labelling occurred at approx. 10 muM-muM-carbamoylcholine. 6. Incubation of muscle fragments with carbamoylcholine provoked a decrease in phosphatidylinositol concentration, as would be expected if phosphatidyl-inositol breakdown is the reaction controlled by agonists. 7. This information all appears consistent with the proposal that phosphatidylinositol breakdown may be a reaction intrinsic to the mechanisms of muscarinic cholinergic receptor systems.

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↗
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

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↗
PubMed1984

Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.

Animals↗
PubMed1985

Properties of receptor-controlled inositol trisphosphate formation in parotid acinar cells.

Activation of muscarinic receptors in rat parotid cells results in breakdown of polyphosphoinositides liberating inositol phosphates, including inositol trisphosphate. Formation of inositol trisphosphate appears independent of agonist-induced Ca2+ mobilization, since neither formation nor degradation of inositol trisphosphate are appreciably altered in low-calcium media, and elevation of cytosolic Ca2+ with a calcium ionophore does not cause an increase in cellular inositol trisphosphate. Further, activation of substance P receptors and alpha 1-adrenoreceptors, but not beta-adrenoreceptors, increases inositol trisphosphate formation. The dose-response curve for methacholine activation of inositol trisphosphate formation more closely approximates the curve for receptor occupancy than for Ca2+-activated K+ release. These results are all consistent with the suggestion that inositol trisphosphate could function as a second messenger linking receptor occupation to cellular Ca2+ mobilization.

Animals↗