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Biomedical subjects

D E Knight

Publications and source records attributed to D E Knight.

At least 55 records · Page 3Linked to original sources
PubMed1986

Observations on the muscarinic activation of catecholamine secretion in the chicken adrenal.

Cells were isolated by collagenase digestion of chicken adrenal glands. Catecholamine secretion could be stimulated by acetylcholine, carbamylcholine, potassium or veratridine. Methacholine, muscarine and oxotremorine were also effective secretagogues whereas nicotine was not. Secretion evoked by acetylcholine was blocked by low concentrations of atropine but was relatively insensitive to hexamethonium. Atropine-sensitive secretion required both external sodium and calcium, was unaffected by tetrodotoxin, blocked by methoxy verapamil and nifedipine, and potentiated by BAY-K-8644. These data suggest that muscarinic activation of these cells facilitates tetrodotoxin insensitive depolarization, thereby opening conventional voltage-sensitive calcium channels. The mechanism by which calcium activates catecholamine secretion was investigated in cells that had been made permeable by exposure to brief intense electric fields. Catecholamine release required Mg-adenosine 5' triphosphate, was half-maximally activated by 1 microM Ca2+ and could be inhibited by high concentrations of Mg2+. At low Ca2+ concentrations, release was potentiated by 12-O-tetradecanoylphorbol 13-acetate, dioctanoylglycerol, guanosine 5'-O-(3-thiotriphosphate) and 5'-guanylylimidodiphosphate, all of which increased the apparent affinity of exocytosis for Ca2+.

Acetylcholine↗
PubMed1985

Effect of various excitatory agonists on the secretion of 5-hydroxytryptamine from permeabilised human platelets induced by Ca2+ in the presence or absence of GTP.

Addition of GTP markedly enhances the ability of thrombin to cause a leftward shift in the Ca2+ dose/response curve for 5-hydroxytryptamine secretion from permeabilised human platelets. Little effect is observed on addition of GTP in the absence of thrombin. Neither ADP nor adrenaline, in the presence or absence of GTP, causes such a shift, whereas 5-hydroxytryptamine does so to a small extent but only in the presence of GTP. The leftward shift in the Ca2+ dose/response curve induced by 12-O-tetradecanoyl-phorbol-13-acetate or 1-oleyl-2-acetylglycerol is not enhanced by addition of GTP. The thrombin concentration required for half-maximal enhancement of the response to Ca2+ is markedly reduced by addition of GTP. The results support the postulate that the effects of excitatory agonists in this system correlate with their ability to activate phospholipase C and provide further evidence for a role for GTP in signal transduction between the receptor and phospholipase C.

Adenosine Diphosphate↗
PubMed1985

The chromaffin granule proton pump and calcium-dependent exocytosis in bovine adrenal medullary cells.

Calcium-dependent exocytosis in 'leaky' bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in 'leaky' cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measured in situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.

Adenosine Triphosphate↗
PubMed1985

Calcium clamp of the intracellular environment.

Quantitative analysis of the effects of calcium on cell function requires methods for altering intracellular free Ca in a precise and reproducible manner. Microinjection of Ca is very unreliable largely because of the powerful Ca-binding properties of cytoplasm. Much more satisfactory are microinjection of Ca-buffers - provided enough buffer is introduced - and various forms of intracellular dialysis and perfusion which permit full equilibration of the cell interior with a defined artificial intracellular environment.

Adrenal Medulla↗
PubMed1984

Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca2+.

Addition of thrombin enhances secretion of both [14C]serotonin and beta-N-acetylglucosaminidase induced by Ca2+ in human platelets rendered permeable by exposure to intense electric fields. Enhancement of beta-N-acetylglucosaminidase secretion by thrombin results from an increase in the maximal extent of the response with no significant change in the median effective concentration EC50 for Ca2+. In contrast, thrombin shifts the dose/response curve for Ca2+-induced [14C]serotonin secretion to the left and has little effect on the maximal extent of this response even when this extent is reduced by use of a non-saturating concentration of MgATP2-. The relationship between extent of response and [MgATP2-] is similar for secretion of [14C]serotonin and of beta-N-acetylglucosaminidase in the presence or absence of thrombin. Similar nucleotide specificities are also observed. Activators of protein kinase C have previously been shown to mimic quantitatively the effect of thrombin on [14C]serotonin secretion induced by Ca2+ [D. E. Knight & M. C. Scrutton (1984) Nature (Lond.) 309, 66-68]. Such activators have the same qualitative effect as thrombin on the properties of beta-N-acetylglucosaminidase secretion induced by Ca2+ but are less effective. The EC50 for thrombin observed for enhancement of [14C]serotonin and beta-N-acetylglucosaminidase secretion is in the same range as that obtained for intact platelets under comparable conditions [D. E. Knight, T. J. Hallam & M. C. Scrutton (1982) Nature (Lond.) 296, 256-257]. The EC50, and the specificity of response, observed for activators of protein kinase C in these systems are consistent with those reported previously for the purified enzyme. Addition of 1-10 microM Ca2+ to permeabilised platelets in the presence of [gamma-32P] ATP causes marked enhancement of 32P incorporation into polypeptides of molecular mass 20 kDA, 45 kDA and 66 kDA. No additional polypeptides become phosphorylated in this system when thrombin is added together with 10 microM Ca2+, but some increase is observed in the extent of phosphorylation of the 45-kDa polypeptide. Addition of 1-oleyl-2-acetylglycerol + 1 - 2 microM Ca2+ causes enhanced phosphorylation of the 45-kDa polypeptide and to a lesser extent of the 20-kDa polypeptide. The dose/response curves for Ca2+-dependent phosphorylation of the 45-kDa polypeptide in the presence and absence of 1-oleyl-2-acetylglycerol are similar to those observed for Ca2+-dependent [14C]serotonin secretion under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylglucosaminidase↗
PubMed1984

Ca2+ and cyclic nucleotide dependence of amylase release from isolated rat pancreatic acinar cells rendered permeable by intense electric fields.

Enzyme digestion of rat pancreatic tissue yielded a preparation of isolated acinar cells, over 90% of which excluded trypan blue. These isolated cells responded to a variety of secretagogues, the responses being sensitive to the removal of extracellular calcium, increasing extracellular magnesium, and by trifluoperazine, an antagonist of Ca-dependent processes. When exposed to intense electric fields, isolated acinar cells became permeable to CaEGTA and MgATP, these markers gaining access to over 60% of the intracellular milieu within minutes. The accessibility to these markers seemed independent of the ionised Ca2+ level. Less than 0.5% of the cellular amylase was released when cells were rendered leaky in a medium containing about 10(-9) M Ca2+, but typically 4% was released when the Ca2+ level was subsequently raised to 10(-5)M levels, the EC50 for Ca2+ being 2 microM. This amount of amylase released was comparable to the amounts secreted from intact cells in response to a variety of agonists. The cytosolic marker lactate dehydrogenase was also released from leaky cells, but the extent was independent of Ca2+ concentration. No amylase was released at 10(-7)M Ca2+ when permeable cells were exposed to cyclic 3',5'-AMP or cyclic 3',5'-GMP. The calcium activation curve for amylase release seemed to be independent of cyclic nucleotides, but was markedly increased in both the extent of release and apparent affinity for Ca2+ in the presence of the phorbol ester 12-0-tetradecanoyl phorbol 13 acetate. These results suggest that when "functionally normal" isolated acinar cells are rendered permeable, Ca2+-but not cyclic nucleotides-acts as a second messenger for amylase secretion, and furthermore that protein kinase C may be involved in the secretory process.

Amylases↗
PubMed1984

Tyrosine hydroxylase in "leaky" adrenal medullary cells: evidence for in situ phosphorylation by separate Ca2+ and cyclic AMP-dependent systems.

The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca2+, the other on cyclic AMP. The Ca2+-dependent system is half-maximally activated by 1-2 microM Ca2+ and 0.5 mM ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 mM) does not inhibit significantly Ca2+-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 microM cyclic AMP and about 0.3 mM ATP. Ca2+-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10(-8)M Ca2+ plus 2 X 10(-5)M cyclic AMP or of 10(-5)M Ca2+, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca2+-dependent system, whereas the other site(s) are phosphorylated by both the Ca2+- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.

Adrenal Medulla↗
PubMed1983

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells.

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.

Acetylcholine↗
PubMed1983

Stimulus-secretion coupling in isolated bovine adrenal medullary cells.

Adrenal medullary cells can be obtained in high yield by protease digestion of slices of bovine adrenal medulla. The properties of catecholamine release from these isolated cells have been determined and compared with the intact perfused gland. Exposure to carbamylcholine, veratridine or high K evokes a transient increase in the rate of catecholamine release in association with dopamine beta hydroxylase but not lactate dehydrogenase. Exposure to A23187 releases catecholamine together with lactate dehydrogenase. In all cases release requires extracellular Ca. A comparison is made of the sensitivities of catecholamine release evoked by various agonists and the effects of nicotinic antagonists, D 600, Na-free media, TTX, Mg and trifluoperazine. Resting and carbamylcholine-evoked catecholamine release is relatively insensitive to the nature of the major extracellular anion and to the external pH over the range 6.4 to 7.8. Measurements with the dye 3 3'-Diethylthiadicarbocyanine iodide (di S-C2-(5)) show that release evoked by carbamylcholine or low concentrations of veratridine is associated--on average--with a small steady depolarization of the cells. Catecholamine release in response to nicotinic agonists, but not the other secretagogues, is greater at 20 degrees C than at 37 degrees C, possibly because of a slower rate of receptor desensitization at this temperature. Transmission electron microscopy of stimulated cells reveals a marked increase in the proportion of large membrane bounded vesicles that lack electron-dense cores. After stimulating secretion in the presence of horseradish peroxidase, the vesicles contain reaction product suggesting that they are generated by endocytosis.

Acetylcholine↗