PubMed Health⌕ Search

Biomedical subjects

P R Dunkley

Publications and source records attributed to P R Dunkley.

At least 37 records · Page 2Linked to original sources
PubMed1996

Oxidative stress and the fetotoxicity of alcohol consumption during pregnancy.

Pregnant Quackenbush Special mice were exposed to ethanol under semiacute (3.0 g/kg body weight intragastrically, days 7 to 12 of pregnancy), and chronic conditions (15% ethanol in drinking water for 5 weeks before and during pregnancy) to assess whether embryo-fetotoxic actions of the drug involve oxidative stress effects. Effects were monitored both in the maternal system and embryo. Alcohol compromised the maternal system by increasing the generation of lipid peroxides in the liver. It also decreased glutathione and vitamin E levels, and glutathione peroxidase and superoxide dismutase activities in this organ. Glutathione peroxidase activity in the maternal blood decreased. Only minor alcohol-induced changes occurred in the uterine endometrium, including decreased xanthine oxidase and increased gamma-glutamyl transpeptidase. Similarly, only few changes were induced in day-12 embryos by alcohol. In this case, glutathione content and xanthine oxidase activity decreased while glutathione reductase activity increased following exposure to the chronic regime. With the possible exception of the maternal liver where evidence of oxidative damage was detected, these results do not reflect substantial changes in the antioxidant defences of either the pregnant mouse or embryo. However, the changes may contribute to the growth retarding and other fetotoxic effects of alcohol when they are totalled into the multifactorial actions of the drug.

Animals↗
PubMed1996

Mn2+ can substitute for Ca2+ in causing catecholamine secretion but not for increasing tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells.

The ability of the divalent cation manganese (Mn2+) to substitute for calcium (Ca2+) both in triggering catecholamine release and in stimulating catecholamine synthesis, as indicated by an increase in tyrosine hydroxylase (TOH) phosphorylation, has been determined in bovine adrenal medullary chromaffin cells maintained in tissue culture. Mn2+ was found to enter chromaffin cells through pathways activated by nicotinic receptor stimulation and potassium depolarisation, and via the Na1:Ca0 exchange mechanism in Na(+)-loaded cells. Like Ca2+, entry of Mn2+ through these pathways triggered immediate catecholamine release and, like Ca2+, maintained quantitatively comparable release at least up to 40 min. Unlike Ca2+, Mn2+ did not stimulate an increase in TOH phosphorylation in intact chromaffin cells, even over a prolonged time course, but Mn2+ did stimulate increased TOH phosphorylation in lysed cell preparations showing that its lack of effect in the intact cells was not due to inhibition of the specific phosphorylation pathway. In lysed cell preparations, Mn2+ stimulated also phosphorylation of a different spectrum of proteins to Ca2+, and of the same proteins to different extents. In particular, P80 (MARCKS protein) was more intensely phosphorylated in the presence of Mn2+ than in the presence of Ca2+. Since TOH phosphorylation always occurs when intracellular Ca2+ is increased, the absence of an increase with Mn2+ indicates that none of its intracellular effects could have occurred as a consequence of Mn2+ mobilisation of intracellular Ca2+. In summary, the data show that Mn2+ is a surrogate for Ca2+ in triggering and maintaining catecholamine release, but does not substitute for Ca2+ in stimulating TOH phosphorylation.

Adrenal Glands↗
PubMed1996

Vitamin A-deficient rats have only mild changes in jejunal structure and function.

This study investigated the effect of clinical and subclinical vitamin A deficiency on intestinal structure and function in rats. Weanling male rats fed a vitamin A-deficient diet (VA-) for 40-42 or 60-63 d were compared with rats either pair-fed (PF) or with free access to the same diet supplemented with vitamin A (VA+). A reference (REF) group was fed a standard rat diet. Weight began to plateau in VA- rats after 42 d, becoming significantly different from PF rats at 60-63 d (P < 0.02). Diarrhea did not develop in any study group. VA- rats had clinical signs of vitamin A deficiency in the 60-63 d study, but not in the 40-42 d study. However, serum and liver retinol concentrations were negligible in all VA- rats. VA- rats in the 60-63 d study had significantly reduced villus height (P < 0.02), and sucrase and maltase activities (P < 0.02) compared with PF rats. There were no differences between VA- and PF rats in mucosal wet weights, protein and DNA concentrations, thymidine kinase activity and glucose transport. No differences were detected in the 40-42 d study for any variable measured. Because clinical vitamin A deficiency in rats causes only mild changes in intestinal structure and function, it is unlikely that these alterations alone are responsible for the interactions observed in epidemiological studies between vitamin A deficiency and diarrheal disease.

Animals↗
PubMed1995

Phosphorylation of synapsin I and dynamin in rat forebrain synaptosomes: modulation by sigma (sigma) ligands.

The effects of sigma (sigma) ligands on protein phosphorylation were examined in crude, rat forebrain synaptosomes. Synaptosomes were prelabelled with 32P(i) and incubated with the sigma ligands 1,3-di-o-tolylguanidine (DTG), (+)pentazocine and (-)pentazocine (3, 10, 30, 100, 300 microM), or haloperidol, reduced haloperidol, and (+)SKF 10,047 (100 microM). Aliquots were then incubated for 10 s in control (5 mM K+) or depolarising buffer (41 mM K+). All the sigma ligands increased basal phosphorylation of synapsin Ib and other proteins including dynamin, and inhibited the depolarisation-dependent increase in phosphorylation of synapsin Ib in synaptosomes. The effects of these ligands are not directly on protein kinases or protein phosphatases. This indicates that the sigma ligands are mediating their effects via interaction with sigma binding sites, and suggest, for the first time, that protein phosphorylation may be one mechanism through which sigma ligands produce their biological effects.

Animals↗
PubMed1995

A role for calcium/calmodulin kinase(s) in the regulation of GABA exocytosis.

A possible role for protein kinases in the regulation of GABA exocytosis in nerve endings was investigated. The effect on the release of the radioactive neurotransmitter ([3H]GABA) from mouse brain synaptosomes of several protein kinase inhibitors was estimated after treatment with 37 mM K+ in the absence of external Na+, a condition under which [3H]GABA release is completely Ca2+ dependent. Among the inhibitors one group inhibit the kinases by the catalytic site (i.e. staurosporine and H7) and others (TFP, sphingosine and W7) act on the regulatory site of protein kinases. The compounds of the second group, which are reported to inhibit calmodulin dependent events and the increase in cytosolic Ca2+ (Cai) induced by high K+ depolarization, were the most efficient inhibitors of [3H]GABA release. The selective inhibitor of CaMPK II, KN-62, also markedly diminished [3]GABA release as well as the increase in Cai induced by high K+. The kinase inhibitors from the first group that are unable to diminish the increase in Cai induced by high K+ were also less efficient inhibitors of [3H]GABA release even at high concentrations. The present results indicate that at the doses tested all the drugs inhibit to some extent the release of the Ca2+ dependent fraction of [3H]GABA perhaps by inhibiting a CaMPK II mediated phosphorylation step triggered by depolarization and facilitated by the elevation of Cai. In addition, the second group of antagonists and KN-62 inhibit the elevation of Cai to high K+ thus exhibiting a higher efficiency on [3H]GABA release than the first group of antagonists.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of intracellular Ca2+ in the histamine H1 receptor-stimulated phosphorylation of Ser8, Ser19, Ser31, and Ser40.

Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H1 receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca2+. In contrast, the H1-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 microM) and significantly reduced by prior exposure to caffeine (10 mM for 10 min) to deplete intracellular Ca2+. Tryptic-phosphopeptide analysis by HPLC revealed that the H1 response in the presence or absence of extracellular Ca2+ resulted in a major increase in the phosphorylation of Ser19 with smaller increases in that of Ser40 and Ser31. In contrast, although a brief stimulation with nicotine (30 microM for 60 s) also resulted in a major increase in Ser19 phosphorylation, this response was abolished in the absence of extracellular Ca2+. These data indicate that the mobilization of intracellular Ca2+ plays a crucial role in supporting H1-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.

Adrenal Medulla↗
PubMed1995

Histamine-stimulated inositol phospholipid metabolism in bovine adrenal medullary cells: a kinetic analysis.

Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [3H]Inositol 1,4,5-trisphosphate [[3H]Ins(1,4,5)P3] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady-state level of approximately 140% of basal values. [3H]Inositol 1,4-bisphosphate [[3H]Ins(1,4)P2] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [3H]inositol 1,3,4-trisphosphate [[3H]Ins(1,3,4)P3], [3H]inositol 1,3-bisphosphate, and [3H]inositol 4-monophosphate ([3H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca2+, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [3H]Ins(1,4,5)P3 was rapidly metabolized (t1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t1/2 values of 113, 133, 104, and 66 s for [3H]Ins(1,3,4)P3, [3H]Ins(1,4)P2, an unresolved mixture of [3H]inositol 1- and 3-monophosphate ([3H]Ins1/3P), and [3H]Ins4P, respectively. The calculated turnover rate of [3H]Ins(1,4,5)P3 was sufficient to account for the turnover of the combination of both [3H]Ins(1,4)P2 and [3H]Ins(1,3,4)P3 but not that of [3H]Ins1/3P or [3H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca(2+)-dependent and -independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5-bisphosphate.

Adrenal Medulla↗
PubMed1993

Synaptosomal amino acid release: effect of inhibiting protein phosphatases with okadaic acid.

The protein phosphatase inhibitor okadaic acid was used to investigate the role of protein phosphatases in regulating the release of amino acids from synaptosomes. Okadaic acid increased the basal release of the amino acids glutamate, aspartate and GABA. The effect was specific in that taurine was not released by either KCl or okadaic acid and there was no synaptosomal lysis or change in ATP/ADP ratios in the presence of okadaic acid. The okadaic acid-stimulated release of amino acids was, however, only a small proportion of that produced by KCl depolarisation. Since okadaic acid raised synaptosomal protein phosphorylation levels to those equivalent to that produced by KCl depolarisation, it is unlikely therefore that there is a direct causal relationship between protein phosphorylation and the release of amino acids. Nevertheless, that release of amino acids from synaptosomes can be elevated under basal conditions by okadaic acid treatment does suggest that okadaic acid-sensitive protein phosphatases have a modulatory role in this process.

Amino Acids↗
PubMed1992

The use of portable blood glucose monitors by trained lay operators.

OBJECTIVE: To evaluate the use of Reflolux II blood glucose reflectance meters by trained lay operators in a community survey. DESIGN: A random household survey involving assessment of participants' blood glucose levels by Reflolux II. For a random subsample of participants an additional sample of blood was placed onto filter paper strips and stored for later examination and assay. SETTING: The survey was conducted in the Newcastle and Lake Macquarie areas of New South Wales. PARTICIPANTS: Of the 1275 participants 15 years and over, 1229 consented to have their blood glucose assessed and 163 of these also had blood stored on filter paper strips. Data were collected by 25 interviewers. MAIN OUTCOME MEASURES: Application of blood spots to filter paper assessed the interviewers' capacity to collect samples of minimal acceptability for Reflolux II operation. Samples were considered adequate if they covered at least a 6 mm soaked disk and there was no evidence that they had been smeared or applied from both sides of the paper. The Reflolux II readings obtained by the interviewers were compared with the blood glucose values from assay of the filter paper samples. RESULTS: Only 63% of the 163 samples collected on filter paper were of acceptable quality for adequate Reflolux II operation. The overall correlation between Reflolux II blood glucose values and those determined from the blood collected on filter paper was good (r = 0.893). However, three of the eight interviewers who had five or more readings for comparison achieved correlation coefficients of < r = 0.20. Of the survey participants identified as having elevated blood glucose levels (> 8 mmol/L) by the filter paper assay, 58% (7 of 12) were not detected by the lay operated reflectance meters. Twenty-two per cent of the individuals assessed by the reflectance meter, compared with 0.5% of a population sample surveyed by the National Heart Foundation, had blood glucose values of less than 3.5 mmol/L. CONCLUSION: It would appear that there may be a considerable error rate in blood glucose values obtained by lay operators using reflectance meters, resulting in underestimation of blood glucose levels. The findings have implications for community awareness programs for diabetes and highlight the need for careful training and monitoring of lay operators in their obtaining of finger-prick blood samples and their use of reflectance meters.

Adolescent↗
PubMed1992

Myelin sheath survival after guanethidine-induced axonal degeneration.

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.

Animals↗
PubMed1992

Tetanus toxin inhibits depolarization-stimulated protein phosphorylation in rat cortical synaptosomes: effect on synapsin I phosphorylation and translocation.

Synapsin I, a prominent phosphoprotein in nerve terminals, is proposed to modulate exocytosis by interaction with the cytoplasmic surface of small synaptic vesicles and cytoskeletal elements in a phosphorylation-dependent manner. Tetanus toxin (TeTx), a potent inhibitor of neurotransmitter release, attenuated the depolarization-stimulated increase in synapsin I phosphorylation in rat cortical particles and in synaptosomes. TeTx also markedly decreased the translocation of synapsin I from the small synaptic vesicles and the cytoskeleton into the cytosol, on depolarization of synaptosomes. The effect of TeTx on synapsin I phosphorylation was both time and TeTx concentration dependent and required active toxin. One- and two-dimensional peptide maps of synapsin I with V8 proteinase and trypsin, respectively, showed no differences in the relative phosphorylation of peptides for the control and TeTx-treated synaptosomes, suggesting that both the calmodulin- and the cyclic AMP-dependent kinases that label this protein are equally affected. Phosphorylation of synapsin IIb and the B-50 protein (GAP43), a known substrate of protein kinase C, was also inhibited by TeTx. TeTx affected only a limited number of phosphoproteins and the calcium-dependent decrease in dephosphin phosphorylation remained unaffected. In vitro phosphorylation of proteins in lysed synaptosomes was not influenced by prior TeTx treatment of the intact synaptosomes or by the addition of TeTx to lysates, suggesting that the effect of TeTx on protein phosphorylation was indirect. Our data demonstrate that TeTx inhibits neurotransmitter release, the phosphorylation of a select group of phosphoproteins in nerve terminals, and the translocation of synapsin I. These findings contribute to our understanding of the basic mechanism of TeTx action.

Animals↗
PubMed1992

Protein phosphorylation in bovine adrenal medullary chromaffin cells: histamine-stimulated phosphorylation of tyrosine hydroxylase.

Histamine can cause the release of catecholamines from bovine adrenal medullary chromaffin cells by a mechanism distinct from that of the depolarizing agents nicotine or high K+ buffer. It was the aim of this study to determine the protein phosphorylation responses to histamine in these cells and to compare them with those induced by depolarization. A number of proteins showed increases in phosphorylation in response to histamine especially when analyzed on two-dimensional polyacrylamide gel electrophoresis or by phosphopeptide mapping; one protein of 20,000 daltons was markedly dephosphorylated. Emphasis was given to the effects of histamine on tyrosine hydroxylase (TOH) phosphorylation, because this protein showed the most prominent changes on one-dimensional gels. Histamine acted via H1 receptors to increase TOH phosphorylation; the response was blocked by the H1 antagonist mepyramine and could be mimicked by the H1 agonist thiazolylethylamine, but not by the H2 agonist dimaprit. The H3 agonist (R) alpha-methylhistamine increased TOH phosphorylation at high concentrations, but the response was blocked entirely by mepyramine. Histamine rapidly increased the phosphorylation of TOH, with a maximum reached within 5 s and maintained for at least 30 min. This was in marked contrast to nicotine-stimulated protein phosphorylation of TOH, which was rapidly desensitized. The initial phosphorylation response to histamine was independent of extracellular Ca2+ for at least 3 min, but the sustained response required extracellular Ca2+. This was in contrast to the situation with both nicotine and high K+ buffer, which under the conditions used here caused a response which was dependent on extracellular Ca2+ at all times investigated. In the presence of histamine, the phosphopeptide profiles for TOH were essentially the same with or without Ca2+, suggesting that the same protein kinases were involved, but at longer times there was evidence of new phosphorylation sites. The mechanism or mechanisms whereby histamine modulates TOH phosphorylation are discussed with emphasis on the differences from depolarizing agents.

Adrenal Medulla↗
PubMed1991

Myelin maintenance by Schwann cells in the absence of axons.

Formation and maintenance of myelin sheaths in the peripheral nervous system are regulated by unknown molecular interactions that are thought to depend upon physical contact between Schwann cells and axons. However, recent studies describing axons surrounded by two concentric myelin internodes in the superior cervical ganglion (SCG) of normal rodents have demonstrated that the outer myelin internodes are maintained without physical contact with the axon. To determine whether the centrally enclosed axon has a trophic effect in maintaining these remote outer internodes, we have produced axonal degeneration by surgical or chemical means. The results indicate that maintenance of myelin internodes totally displaced from axonal contact depends neither upon the presence of the axon nor on diffusible axonal factors. A further implication of these studies is that myelin breakdown during Wallerian degeneration is regulated by a positive signal which originates in degenerating nerves, rather than solely by loss of axonal trophic substances.

Animals↗
PubMed1991

Modulation of synaptosomal protein phosphorylation/dephosphorylation by calcium is antagonised by inhibition of protein phosphatases with okadaic acid.

The protein phosphatase inhibitor okadaic acid was used to investigate the protein phosphatases involved in the endogenous dephosphorylation of proteins in intact synaptosomes. Despite the fact that the calcium-dependent protein phosphatase (calcineurin) is most concentrated in synaptosomes and accounts for approximately 0.3% of synaptoplasmic protein, the majority of the dephosphorylation activity under both basal and depolarisation conditions is due to protein phosphatase type 1 (PP1) and/or protein phosphatase type 2A (PP2A). Nevertheless our results do suggest that calcineurin is active in synaptosomes and has 2 effects: a rapid, direct dephosphorylation of a limited range of substrates and an indirect activation of PP1 presumably by dephosphorylation of protein phosphatase 1 inhibitor-1.

Animals↗
PubMed1991

Opioid inhibition of nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells.

The ability of a number of opioid agonists and antagonists to affect nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells has been investigated. High (10 microM) concentrations of the opioid agonist bremazocine produced a significant inhibition of nicotine-induced 45Ca2(+)-uptake throughout the 15 min time course examined. The opioid subtype-selectivity of this inhibition was investigated; mu and delta selective agonists produced only minor effects whereas the kappa selective agonist U50-488H and the endogenous opioid peptides dynorphin(1-13) and metorphamide almost abolished nicotine-induced 45Ca2(+)-uptake. The U50-488H inhibition was significant at 10 nM concentrations with an IC50 of approximately 1 microM. U50-488H inhibition could not be reversed or reduced by the opioid antagonists naxolone, diprenophine or Mr2266. Furthermore, Mr2266 and its optical isomer Mr2267 also produced marked inhibition of 45Ca2(+)-uptake. The inhibition was specific to nicotine-induced 45Ca2(+)-uptake in that a similar level of uptake evoked by potassium depolarization was unaffected by high concentrations of U50-488H. These data indicate that opioid inhibition of nicotine-induced 45Ca2(+)-uptake does not involve classical, stereospecific opioid receptors and suggests the involvement of a pharmacologically distinct opioid recognition site. It is speculated that this may be associated with the nicotine receptor-ionophore complex.

Adrenal Medulla↗
PubMed1991

Autophosphorylation of neuronal calcium/calmodulin-stimulated protein kinase II.

A unique feature of neuronal calcium/calmodulin-stimulated protein kinase II (CaM-PK II) is its autophosphorylation. A number of sites are involved and, depending on the in vitro conditions used, three serine and six threonine residues have been tentatively identified as autophosphorylation sites in the alpha subunit. These sites fall into three categories. Primary sites are phosphorylated in the presence of calcium and calmodulin, but under limiting conditions of temperature, ATP, Mg2+, or time. Secondary sites are phosphorylated in the presence of calcium and calmodulin under nonlimiting conditions. Autonomous sites are phosphorylated in the absence of calcium and calmodulin after initial phosphorylation of Thr-286. Mechanisms that lead to a decrease in CaM-PK II autophosphorylation include the thermolability of the enzyme and the activity of protein phosphatases. A range of in vitro inhibitors of CaM-PK II autophosphorylation have recently been identified. Autophosphorylation of CaM-PK II leads to a number of consequences in vitro, including generation of autonomous activity and subcellular redistribution, as well as alterations in conformation, activity, calmodulin binding, substrate specificity, and susceptibility to proteolysis. It is established that CaM-PK II is autophos-phorylated in neuronal cells under basal conditions. Depolarization and/or activation of receptors that lead to an increase in intracellular calcium induces a marked rise in the autophosphorylation of CaM-PK II in situ. The incorporation of phosphate is mainly found on Thr-286, but other sites are also phosphorylated at a slower rate. One consequence of the increase in CaM-PK II autophosphorylation in situ is an increase in the level of autonomous kinase activity. It is proposed that the formation of an autonomous enzyme is only one of the consequences of CaM-PK II autophosphorylation in situ and that some of the other consequences observed in vitro will also be seen. CaM-PK II is involved in the control of neuronal plasticity, including neurotransmitter release and long-term modulation of postreceptor events. In order to understand the function of CaM-PK II, it will be essential to ascertain more fully the mechanisms of its autophosphorylation in situ, including especially the sites involved, the consequences of this autophosphorylation for the kinase activity, and the relationships between the state of CaM-PK II autophosphorylation and the physiological events within neurons.

Animals↗