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D E Knight

Publications and source records attributed to D E Knight.

At least 37 records · Page 2Linked to original sources

Tissue selectivity of endothelin.

The effects of endothelin, a potent endogenous vasoconstrictor peptide, were examined in a range of vascular and non-vascular tissues. At concentrations that cause vasoconstriction in portal vein and aorta, the peptide strongly contracted rat uterus, trachea and vas deferens, but not guinea pig ileum. Nifedipine, a dihydropyridine calcium anatgonist, partially inhibited these contractions. Endothelin had no inotropic or chronotropic effect on the isolated rat heart. The peptide did not modulate secretion at the neuromuscular junction, from adrenal medullary cells or neutrophils, nor affect secretion or aggregation of platelets. The tissue responsiveness to endothelin was not the same as the tissue distribution of dihydropyridine receptors. This supports the idea that endothelin interacts with a specific receptor distinct from dihydropyridine sensitive calcium channels. The contractile effect of endothelin on non vascular smooth muscle suggests that the concept of endothelium dependent modulation of vascular smooth muscle tone may be extended to include epithelium dependent modulation of non vascular tissues.

Animals↗

A method for making solution changes in the sub-millisecond range at the tip of a patch pipette.

A method is described here for making multiple fast external solution changes at the tip of a patch pipette. The time for the change, 0.2 ms, has been established by measuring changes in liquid junction potential at the tip of an open patch pipette. This technique of producing an abrupt change in solution allows agonist/receptor reactions to be studied under non-equilibrium conditions. We have applied this technique to the nicotinic receptors in outside-out patches from skeletal muscle cell line C2 (Jaffe and Saxel 1979) and from bovine adrenal chromaffin cells. The application of step changes in acetylcholine concentration produces current traces with a characteristic shape, which may be compared with the predictions of established models for the activation and desensitisation of the nicotinic receptor. The results of making single steps and also short pulses in acetylcholine concentration are demonstrated. The direct comparison of two different cholinergic agonists is demonstrated.

Acetylcholine↗

Activation of sodium channels is not essential for endothelin induced vasoconstriction.

The role of sodium and calcium ions in the vasoconstrictor response of isolated rat aorta and protal vein to synthetically prepared endothelin is investigated. Contractile responses to endothelin, unlike those induced by the sodium channel activator veratridine, are unaffected by tetrodotoxin or by the removal of sodium chloride from the solution bathing the tissue. The responses are the same whether sodium chloride is replaced iso-osmotically with either sucrose or potassium chloride. The endothelin responses in all media are entirely dependent on the presence of extracellular calcium, and can be blocked by 1 microM nitrendipine. These findings offer no support to the idea that voltage activated sodium channels are the primary site of action of endothelin as suggested by sequence homologies to scorpion alpha-toxins, but are entirely consistent with the possibility that the site of action of endothelin is closely coupled to the calcium channel (Yanagisawa et al, 1988).

Animals↗

Calcium-dependent and calcium-independent exocytosis.

A large body of evidence supports the concept that calcium (Ca2+) plays a pivotal role in the control of exocytosis. However, recent experiments suggest that a rise in intracellular Ca2+ does not necessarily trigger secretion, and also that secretion can occur independently of cytosolic free calcium levels. This article briefly summarizes the early evidence that has formulated the role of Ca2+ in secretion, and then examines some of the recent evidence suggesting a Ca2+-independent mechanism of exocytosis.

Animals↗

Ionic requirements of the endothelin response in aorta and portal vein.

The vasoconstrictor responses of isolated rat portal vein and aorta to synthetically prepared endothelin are investigated. Both preparations respond to 10(-9) M levels of the peptide although the aortic response is more sustained than that of the portal vein. Endothelin-evoked contractions, unlike those evoked by scorpion alpha-toxins (which are homologous to endothelin) or by veratridine, are insensitive to tetrodotoxin or to the removal of sodium ions from the tissue-bathing medium. Contractile responses to endothelin may still be observed in high-potassium depolarizing medium and are not dependent on the presence of extracellular chloride; however, the responses are dependent on the presence of extracellular calcium and are blocked by nitrendipine, nifedipine, or nickel. Endothelin-evoked uptake of 45Ca into aortic tissue is also independent of extracellular sodium or potassium and is blocked by nifedipine. These data strongly suggest that endothelin acts at a site closely coupled to the calcium channel and that depolarization by sodium influx through voltage-dependent channels is not involved in endothelin-induced vasoconstriction.

Animals↗

Botulinum toxin-induced ADP-ribosylation and inhibition of exocytosis are unrelated events.

The hypothesis that inhibition of secretion by botulinum neurotoxin type D occurs by an intracellular process involving ADP-ribosylation has been directly tested by measuring both the extent of inhibition of secretion and of ADP-ribosylation in the same cells. Although the inhibitory effect of unpurified toxin closely parallels intracellular ribosylation, the two events are clearly unrelated, as using purified D and C3 toxins together with their antibodies, each of these events can be either stimulated or inhibited independently of each other.

Adenosine Diphosphate Ribose↗

Evidence implicating protein kinase C in exocytosis from electropermeabilized bovine chromaffin cells.

The calcium sensitivity of exocytosis from electro-permeabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electro-permeabilized cells are exposed to Ca2+ levels much in excess of 50 microM. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca2+ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.

Animals↗

Stimulus-secretion coupling: a perspective highlighting the contributions of Peter Baker.

Many investigators are using numerous preparations for contributing to our present understanding of stimulus-secretion coupling, by which we mean stimulus-dependent exocytosis, sometimes known as the regulated pathway. However, a few model systems have been particularly illuminating and several of these were exploited by Peter Baker and his close associates: namely, the motor nerve terminal, the adrenal chromaffin cell, the sea urchin egg and the blood platelet. In fact, Peter's first real contribution in this area came from his seminal studies on calcium transport in his favourite preparation, the squid giant axon, where he investigated Ca2+/Na+ exchange, Ca2+ distribution and voltage-gated Ca2+ entry. More direct investigations into stimulus-secretion coupling came from work on neurone transmitter release in collaboration with Andrew Crawford, and on catecholamine secretion from the adrenal medulla in collaboration (with TJR). His most important generic contribution to this field was in the development (with DEK), of the electropermeabilized cell, which allows control of the low molecular weight components of the cytosol while leaving the exocytotic apparatus and process intact. In the initial experiments on the cells it was finally proved that Ca2+-dependent secretion of catecholamines is indeed from the granules and not from the cytosol. The quantification of the Ca2+ requirement of secretory exocytosis was an important step, as was the investigation of many factors purported to be important in the coupling mechanism or in the exocytotic process itself. Work with the human platelet, using this technique, has proved to be especially valuable in unravelling the complex interactions between different second messengers and has been neatly complemented by work in intact cells containing Ca2+-indicator fluorescent dyes. Peter was also intrigued by post-secretory events both in the early seventies, and at the end of his career when he embarked on analysis of the membrane retrieval process and the associated uptake of extracellular medium.

Animals↗

Secretion of 5-hydroxytryptamine from electropermeabilised human platelets. Effects of GTP and cyclic 3',5'-AMP.

Enhancement by thrombin of Ca2+-dependent 5HT secretion in the absence of added GTP decreases as the time between electropermeabilisation and addition of thrombin is increased. No decrease occurs if thrombin is added with GTP. Observation of apparent GTP-independent receptor/phospholipase C coupling may result from the presence of bound GTP in the preparation. Enhancement by GTP of Ca2+-dependent 5HT secretion occurs with a significant lag indicating an agonist-independent effect. Cyclic 3'5'-AMP inhibits enhancement by GTP of Ca2+-dependent 5HT secretion while having no effect on enhancement induced by GTP gamma S. Hence cyclic AMP may impair receptor/phospholipase C coupling by enhancing Np GTPase activity.

Blood Platelets↗

Calcium and diacylglycerol control of secretion.

Measurements of intracellular Ca2+ in adrenal medullary cells suggest that a transient rise in Ca2+ leads to a transient secretory response, the rise in Ca2+ being brought about by an influx through voltage-sensitive Ca channels which subsequently inactivate. The level of Ca2+ observed is much smaller than the Ca2+ needed to trigger secretion when introduced directly into the cell. The discrepancy is removed by the presence of diacylglycerol, which increases the sensitivity of the secretory process to Ca2+. The site of action of Ca2+ and diacylglycerol is probably protein kinase C, and the different secretory responses to increases of Ca2+ and diacylglycerol can be modelled in terms of a preferential order of binding of these two substrates to the enzyme. ATP is needed for secretion: one role is possibly to confer stability to the secretory apparatus; another may involve phosphorylation of some key protein. The kinetics of secretion suggest that if Ca2+ regulates phosphorylation or dephosphorylation, then it is the rate of change of phosphorylation that controls secretion rather than the extent of phosphorylation or dephosphorylation. Guanine nucleotide-binding proteins may play a role not only at the level of signal transduction coupling, but also at or near the site of exocytosis, and the mechanism by which some Botulinum toxins inhibit secretion may be associated with these proteins.

Adrenal Glands↗

Botulinum toxin types A, B and D inhibit catecholamine secretion from bovine adrenal medullary cells.

Evoked catecholamine secretion from cultured bovine adrenal medullary cells is inhibited by commercially available botulinum toxins - types A, B and D (10(4)-10(6) MLD/ml of culture medium). Basal secretion is also inhibited. The catecholamine content of such toxin-treated cells is larger than that of control cells and may in part be a result of the inhibition of basal release. The onset of action of botulinum toxin types A and D can be neutralised by their respective antisera. Concentrations of botulinum toxins A, B or D that inhibit secretion leave unaffected the 45Ca2+ influxes normally associated with secretion. These data provide further evidence to support the idea [(1985) Nature 317, 719-721] that botulinum toxins block secretion by acting downstream of the Ca2+ transient at or near the site of exocytosis.

Adrenal Medulla↗

Effects of guanine nucleotides on the properties of 5-hydroxytryptamine secretion from electropermeabilised human platelets.

Guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta,gamma-imido]triphosphate enhance Ca2+-dependent 5-hydroxytryptamine secretion from electropermeabilised human platelets. GTP has little such effect except when the platelets are permeabilised, and incubated with this nucleotide, at 2 degrees C and pH 7.4. The lag phase observed in the time course of 5-hydroxytryptamine secretion induced by addition of guanosine 5'-[gamma-thio]triphosphate is markedly longer than that characterising secretion induced by Ca2+ alone, by thrombin +/- GTP or by guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin. GTP causes competitive inhibition of the enhancement of the Ca2+-dependent secretory response induced by guanosine 5'-[gamma-thio]triphosphate when both nucleotides are added simultaneously. The extent of inhibition is decreased if guanosine 5'-[gamma-thio]triphosphate is added prior to GTP. GTP markedly enhances the effect of thrombin on Ca2+-dependent 5-hydroxytryptamine secretion by increasing the maximal extent of the response and decreasing the thrombin concentration required to give half-maximal response. A similar effect is observed on addition of guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin at short incubation times. On more prolonged incubation the effects of thrombin and guanosine 5'-[gamma-thio]triphosphate are additive. Guanosine 5'-[beta-thio]diphosphate completely inhibits the response induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma-imido]triphosphate but has little effect on the response induced by Ca2+ when added alone or in the presence of thrombin. Partial inhibition is observed for the response induced by thrombin + GTP. Cyclic-AMP effectively inhibits the response induced by thrombin + GTP but has little effect on that induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma]imidotriphosphate. The results provide further support for the proposal [Haslam, R.J. & Davidson, M.M.L. (1984) FEBS Lett. 170, 90-95], that receptor--phospholipase-C coupling in platelets is mediated in part by a guanine-nucleotide-binding (Np) protein but that a coupling mechanism may also exist which is independent of such a protein. The properties of guanine-nucleotide-dependent coupling resemble those previously described for receptor--adenylate-cyclase coupling.

Blood Platelets↗

Calcium and exocytosis.

Amine secretion from electropermeabilized bovine chromaffin cells and human platelets requires Ca2+ and MgATP. There appears to be little correlation between the pH or potential of the interior of the amine storage granules of the chromaffin cells and the Ca2+ sensitivity or extent of secretion. The Ca2+ sensitivities of secretion for both preparations are increased by activators of protein kinase C. In the platelet, thrombin also increases the Ca2+ sensitivity. The thrombin-induced response is further enhanced by micromolar levels of GTP. The non-hydrolysable analogue GTP gamma S also potentiates the Ca2+-dependent secretory response, but this effect is additive to that seen by thrombin rather than synergistic, as is the case with GTP. GTP gamma S inhibits catecholamine secretion from bovine chromaffin cells. In both preparations the effects of GTP gamma S are inhibited by 10 microM GTP, even though GTP concentrations up to 1 mM are without effect when added alone. These results are consistent with there being two sites of action for the guanine nucleotides, one at the level of the agonist receptor and activated by GTP or one of its breakdown products, and the other one activated by GTP gamma S--possibly at the level of protein kinase C itself.

Adenosine Triphosphate↗