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PubMed1989

Sanguinarine, a phototoxic H2O2-producing alkaloid.

Sanguinarine chloride, a quaternary salt of a benzophenanthrene alkaloid, was phototoxic to catalase-deficient strains of Escherichia coli but not to Trichoplusia ni (cabbage looper moth larvae), an insect with high levels of catalase activity. Chemical analyses confirm that sanguinarine is an efficient producer of H2O2. This differential toxicity suggests that the mode of phototoxic action involves production of H2O2 which could be detoxified in many organisms by catalase.

Alkaloids↗
PubMed1992

Photophysical property of sanguinarine in the excited singlet state.

The photophysical property of the alkanolamine form of sanguinarine has been studied in aqueous and organic medium under various environmental conditions from the measurement of absorption, fluorescence and NMR spectroscopy. Alkanolamine fluorescence shows an excitation time dependent fluorescence quenching and the rate of quenching increases significantly with increasing pH and concentration of the species, while it decreases with increasing temperature. This phenomenon is explained by excited state intramolecular proton transfer from a 6-OH group to the lone pair of nitrogen through the formation of zwitterion.

Alkaloids↗
PubMed1992

Phototoxic and photochemical properties of sanguinarine.

Sanguinarine, a commercial drug exhibiting antimicrobial and antitumor properties, was studied with respect to its basic photochemical characteristics and also with regard to its phototoxicity to mosquito larvae (Aedes atropalpus). Sanguinarine proved to be clearly phototoxic to larvae, with an LD50 of 0.096 mg/mL with near UV exposure as compared with 23.3 mg/mL without. Flash photolysis experiments enabled the study of the triplet state of sanguinarine to be undertaken. Quenching by oxygen occurs with a rate constant of 6 x 10(9) M-1s-1 and time-resolved emission studies indicate that sanguinarine produces a significant amount of singlet oxygen (phi delta = 0.16) as does the isoquinoline alkaloid, berberine (phi delta = 0.25). These values represent the first direct quantitative measurements of photosensitization parameters of these compounds. Additionally, sanguinarine exhibits efficient electron donation properties, undergoing reaction with methyl viologen with a rate constant greater than 10(10) M-1s-1, but is a poor electron acceptor. Phototoxicity of sanguinarine can thus be explained in terms of its photosensitization properties.

Aedes↗
PubMed2002

The association between Viadent use and oral leukoplakia--results of a matched case-control study.

OBJECTIVES: Several oral pathologists have described oral leukoplakia of the maxillary vestibule in patients with no traditional risk factors for the condition. On questioning these patients, it was determined that Viadent mouthrinse or Viadent toothpaste was commonly used by them. A hypothesis was developed that Viadent or a component of Viadent caused the lesions. This paper evaluates the association between oral leukoplakia and use of Viadent products. METHODS: A matched case-control study was designed to test the hypothesis that use of Viadent products increases an individual's risk of oral leukoplakia. Cases included 58 patients diagnosed with oral leukoplakia identified through the biopsy service at the Ohio State University, College of Dentistry, Oral Pathology Section. The matched control was a friend or relative of the patient. Cases and controls were administered a questionnaire about their use of Viadent, and other known risk factors for leukoplakia such as tobacco and excessive alcohol use. RESULTS: An age difference was seen between cases and controls, the cases being older (P < .001). After controlling for confounding factors, results of exact conditional logistic regression analyses showed that use of Viadent products was a risk indicator for oral leukoplakia (odds ratio = 10.0; 95% confidence interval = 2.0, 89.2). CONCLUSIONS: Viadent use is a risk indicator for oral leukoplakia, confirming our previous findings.

Alcohol Drinking↗
PubMed1997

Efficacy of a dentifrice and oral rinse containing sanguinaria extract in conjunction with initial periodontal therapy.

In the treatment of periodontal disease initial therapy aims at reducing marginal inflammation so allowing assessment of residual disease and further treatment options. The aim of the present study was to determine whether the use of a dentifrice and oral rinse containing sanguinaria extract led to a more rapid resolution of gingival inflammation following initial therapy. Thirty-four subjects, randomly assigned to one of two treatment groups, took part in this randomized double-blind parallel study. All subjects received initial therapy including oral hygiene instruction and scaling and root planing as required. One group also received an active dentifrice and oral rinse containing sanguinaria extract (an antiplaque agent) and zinc chloride. The other group received a placebo dentifrice and oral rinse. The gingival index (GI), plaque index (PLI) and probing pocket depths (PD) were recorded at six sites per tooth at baseline, two weeks after initial therapy and six weeks after initial therapy. There was no significant difference between the groups for any of the parameters at the baseline examination. Two weeks following initial therapy both groups showed a statistically significant increase in the number of sites with PLI of 0 or 1 (p < 0.0001) and a statistically significant increase in the number of sites with a GI of 0 or 1 (that is, no bleeding on probing), (p < 0.0001). Also there was a statistically significant increase in the number of sites with probing depths < or = 3 mm (p < 0.0001) compared with baseline. These changes were maintained through to six weeks post therapy. There was no significant advantage to the sanguinaria group. Results demonstrate that initial therapy in the form of oral hygiene instruction, scaling and root planing leads to a significant improvement in periodontal status which is maintained at least in the short term. Further, use of a dentifrice and oral rinse containing sanguinaria did not improve the efficacy of initial therapy.

Adult↗
PubMed2005

Effects of sanguinaria in fluoride-containing dentifrices on the remineralisation of subsurface carious lesion in vitro.

OBJECTIVE: The purpose of this study was to investigate the potential cariostatic benefits of dentifrice containing both fluoride (F) and sanguinaria (SG) in a silica abrasive formula using the microbial de- and re-mineralisation cycle model. METHODS: Samples of bovine tooth enamel were embedded in resin, polished, exposed to a lactate carbopol buffer system for 44 hours, and divided into one control and five test groups of 12 specimens each by randomised blocks with VHN stratification. Surface microhardness was compared before and after the microbial cyclic sequences. RESULTS: VHNs for groups treated with dentifrices containing 1,000 ppm F from MFP with or without bamboo salt (BS) were significantly higher than the control. The groups treated with dentifrices containing F from NaF/silica with or without SG had significantly higher VHNs than that with F from MFP/DCPD. The VHNs treated with dentifrice containing both NaF-SG demonstrated the highest among test groups, indicating that there may be a synergistic effect in remineralising the incipient subsurface lesion. CONCLUSION: The dentifrice containing both NaF and SG showed a significantly greater effect (p < 0.05) on the remineralisation of the enamel lesion than the other test dentifrices.

Alkaloids↗
PubMed1998

Protein kinase inhibitors attenuate cardiac swelling-induced amino acid release in the rat.

Rat Langendorff heart preparations have been used to study the efflux of cardiac amino acids into coronary artery perfusates during brief (5-min) periods of exposure to hyposmotic stress (70 mM NaCl). Coronary flow rates, heart rates and intra-aortic pressures were recorded. Amino acid levels were measured by high-performance liquid chromatography. Hyposmotic stress caused marked percentage increases in taurine, glutamate and aspartate levels in the coronary perfusate, with smaller increases in phosphoethanolamine, glycine and alanine and non-significant increases in serine and glutamine. Amino acid levels declined during reperfusion with isosmotic Krebs-Henseleit bicarbonate buffer. Inhibition of protein kinase C with chelerythrine chloride (5 microM) depressed the osmotically-induced release of aspartate, glutamate, taurine and glycine. The protein tyrosine kinase inhibitor, genistein, reduced the anisosmotic efflux of aspartate, glutamate, taurine and phosphoethanolamine. Lavendustin A, another inhibitor of tyrosine kinase, depressed the osmotically evoked release of aspartate, glutamate and taurine. These studies demonstrate the involvement of protein kinase C and tyrosine kinases in the efflux of amino acids from the osmotically challenged rat heart and imply that these enzymes are involved in the mechanisms responsible for volume regulation by cardiac cells.

Alkaloids↗
PubMed1979

Activation by sanguinarine of active sodium efflux from frog skeletal muscle in the presence of ouabain.

1. Applied to intact Na-rich muscle cells, sanguinarine causes an increased 22Na efflux in the presence or absence of extracellular K+ or of ouabain. 2. The increased 22Na efflux does not represent Na:Na exchange as indicated by the fact that it is not associated with an increase in one-way isotopic Na influx nor is it abolished by the absence of external Na+. 3. In both K-free Ringer and K-free Ringer containing ouabain, sanguinarine not only increases one-way efflux of 22Na, it also induces net efflux of Na+ in the face of both an electrical and a concentration gradient. Moreover, the induction of net Na+ efflux occurs in the face of an approximately fourfold increase in PNa. These surprising results lead to the conclusion that, contrary to all experiments, sanguinarine induces active Na+ efflux even in K-free Ringer containing 10(-3) M-ouabain. 4. Sanguinarine depolarizes the Na-loaded muscle to approximately the same value, -54 mV, regardless of the presence or absence of extracellular K+. This depolarization is most likely secondary to the increase in PNa. 5. Sanguinarine causes a net loss of K+, presumably secondary to the depolarization. 6. The stimulation of net Na+ efflux is not correlated with the depolarization. The stimulation in K-free conditions (with or without ouabain), which is associated with the largest depolarization, produces an increment in net Na+ efflux which is not significantly different from the increment in net Na+ efflux in 10 mM-K+ Ringer where the depolarization is smallest. 7. Although sanguinarine increases active Na+ efflux in intact cells, it inhibits the isolated (Na+ + K+)-ATPase, presumably due to interaction with a site on the inner face of the membrane fragment. 8. The surprising stimulation of active Na+ efflux in the presence of 10(-3) M-ouabain must be due to interaction of sanguinarine with a site on the outer face of the membrane, perhaps the K+ activation site. It seems probable that the component of active Na+ efflux induced by sanguinarine is mediated by the Na pump. Sanguinarine may produce a K+-like effect upon the Na pump with consequent unbinding of ouabain.

Alkaloids↗
PubMed1996

Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes.

1. The effect of angiotension II (Ang) on delayed rectifier K+ current (IK(V)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole-cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole-cell IK(V) was assessed in myocytes dialysed with 10 mM BAPTA, 5 mM ATP and 1 mM GTP either at room temperature or at 30 degrees C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug. Tail current recorded after 250 ms pulses to +30 mV and repolarization to -40 mV was reduced from 3.9 +/- 0.7 to 2.5 +/- 0.5 pA pF-1 at 20 degrees C (n = 6) and from 4.5 +/- 0.5 to 3.13 +/- 0.4 pA pF-1 at 30 degrees C(n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 microM), which alone had no direct effect on the amplitude of IK(V). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 microM intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (tau 1 reduced from 377 +/- 32 to 245 +/- 11 ms; tau 2 unchanged, n = 17). Neither the voltage dependence of activation nor inactivation were affected by Ang. 5. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKC inhibitors, calphostin C (1 microM) and chelerythrine (50 microM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme-specific antibodies: alpha, epsilon and zeta isoenzymes were detected, but beta, gamma, delta and eta isoenzymes were not. 7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca(2+)-independent PKC isoenzyme epsilon in the signal transduction pathway responsible for IK(V) inhibition by Ang.

Alkaloids↗
PubMed1997

Alpha 1-adrenoceptor activation of a non-selective cation current in rabbit portal vein by 1,2-diacyl-sn-glycerol.

1. The transduction mechanisms involved in the activation and modulation of the noradrenaline-activated cation current (Icat) were investigated with whole-cell patch clamp techniques in rabbit portal vein smooth muscle cells. 2. Intracellular application of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S, 500 microM) evoked a 'noisy' inward current at -50 mV with a similar current-voltage relationship and reversal potential to the current evoked by bath application of noradrenaline (100 microM). Guanosine 5-O-(2-thiodiphosphate) (GDP beta S, 1 mM) markedly inhibited noradrenaline-activated Icat. 3. The phospholipase C (PLC) inhibitor U73122 inhibited the amplitude of the noradrenaline-activated Icat in a concentration- and time-dependent manner and the IC50 was about 180 nM. U73122 had similar effects on the cation current evoked by GTP gamma S. 4. Intracellular application of myo-inositol 1,4,5-trisphosphate (IP3, 100 microM) from the patch pipette did not activate any membrane current in cells where intracellular calcium concentration ([Ca2+]i) was buffered to 14 nM, but subsequent addition of noradrenaline evoked Icat. 5. Bath application of the 1,2-diacyl-sn-glycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10 microM) activated Icat, whereas the phorbol ester phorbol 12,13-dibutyrate (PDBu, 0.1-5 microM) failed to activate Icat, in every cell examined. Icat activated by OAG after bath application of PDBu was not significantly different from OAG-activated Icat in the absence of PDBu. The DAG lipase inhibitor RHC80267 (10 microM) activated Icat in some cells, whereas the DAG kinase inhibitor R59949 (10 microM) never activated Icat. 6. Bath application of the protein kinase C inhibitor chelerythrine (1-10 microM) had no effect on either OAG-or noradrenaline-activated Icat. 7. It is concluded that noradrenaline activates Icat via a G-protein coupled to PLC and that the resulting DAG product plays a central role in the activation of cation channels via a protein kinase C-independent mechanism.

Alkaloids↗
PubMed2002

Voltage independence of vasomotion in isolated irideal arterioles of the rat.

The cellular mechanisms underlying vasomotion of irideal arterioles from juvenile rats have been studied using electrophysiological methods, ratiometric calcium measurements and video microscopy. Vasomotion was not affected by removal of the endothelium. Spontaneous contractions were preceded by spontaneous depolarizations. Both were abolished by the intracellular calcium chelator, BAPTA AM (20 microM), but not by ryanodine (10 microM), suggesting a dependence on the cyclical release of calcium from intracellular stores, other than those operated by ryanodine receptors. Oscillations were little changed when the membrane potential of short segments of arteriole was either depolarized or hyperpolarized. When the segments were voltage clamped, oscillating inward currents were recorded, indicating that the changes in membrane potential were voltage independent. Vasomotion was preceded by intracellular calcium oscillations and both were abolished by inhibitors of phospholipase C (U73122, 10 microM), phospholipase A(2) (AACOCF(3), 30 microM) and protein kinase C (chelerythrine chloride, 5 microM, and myristoylated protein kinase C peptide, 10 microM). Inhibition of vasomotion by the dual lipoxygenase and cyclo-oxygenase inhibitor, NDGA (10 microM), the lipoxygenase inhibitor, ETI (1 microM) but not by the cyclo-oxygenase inhibitors, aspirin (10 microM) and indomethacin (10 microM), or the cytochrome P450 inhibitor 17-ODYA (10 microM), suggested an involvement of the lipoxygenase pathway. The observations suggest that vasomotion of iris arterioles is voltage independent and results from the cyclical release of calcium from IP(3)-sensitive stores which are activated by cross talk between the phospholipase C and phospholipase A(2) pathways in vascular smooth muscle.

Alkaloids↗
PubMed2002

Modulation of volume-sensitive chloride current by noradrenaline in rabbit portal vein myocytes.

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Excitation of smooth muscles isolated from the guinea-pig gastric antrum in response to depolarization.

In small segments of circular smooth muscle bundle isolated from the guinea-pig gastric antrum, depolarization of the tissue with intracellular current stimuli evoked regenerative slow potentials after a refractory period of 5-10 s. The refractory period changed inversely with the amplitude and duration of the stimulating depolarization. Thapsigargin (an inhibitor of calcium-ATPase at internal stores), 2-aminoethoxydiphenyl borate (2-APB, an inhibitor of inositol 1,4,5-trisphosphate (IP3)-receptor-mediated Ca2+ release), and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) reduced the amplitude of slow potentials, with no significant alteration of the refractory period. Bisindolylmaleimide I or chelerythrine (inhibitors of protein kinase C, PKC) increased the refractory period and inhibited the amplitude of slow potentials. These results indicate that the refractory period and amplitude of slow potentials are related to the activation of PKC and the amount of Ca2+ released from the internal stores through activation of IP3 receptors, respectively. Acetylcholine (ACh) reduced the refractory period and increased the amplitude of slow potentials: the former was antagonized by chelerythrine and the latter by 2-APB. The results suggest that ACh has dual actions; stimulation of the metabolism of inositol phosphate and activation of PKC. Phorbol-12-myristate-13-acetate, a selective stimulant of PKC, at low concentrations (< 10 nM) mimicked the actions of ACh and at high concentrations reduced the frequency of slow potentials and increased the refractory period. The possible involvement of the concentration-dependent differences in the actions of phorbol ester on the translocation of PKC was considered.

Acetylcholine↗
PubMed2003

Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA.

Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA' mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5-10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA.

Alkaloids↗
PubMed2003

Properties of a constitutively active Ca2+-permeable non-selective cation channel in rabbit ear artery myocytes.

In smooth muscle, non-selective cation conductances contribute to agonist-evoked depolarisation and contraction, and in the present study using patch-pipette techniques we describe the properties of a constitutively active cation channel. With whole-cell recording in K+-free conditions, there was a spontaneous current with a reversal potential (Er) that was altered by replacement of external Na+ by an impermeant cation, but not when external Cl- was replaced by an impermeant anion. The tonic cation inward current could be carried by Ca2+ ions and was greatly enhanced when the external Ca2+ concentration was reduced. In outside-out patches there was spontaneous cation channel activity that could be resolved into three conductance states of about 15, 25 and 40 pS, all with the same Er as the whole-cell current. Kinetic analysis revealed that there were two open times of about 1 and 5 ms and that the currents displayed bursting kinetics with burst durations of approximately 5 ms and 25 ms. Removal of external Ca2+ ions increased the probability of channel opening (Po) sixfold, which was associated with an increase in the longer burst duration. Bath application of the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol increased Po, but phorbol 12,13-dibutyrate, which stimulates protein kinase C (PKC), reduced channel activity. In contrast, the PKC inhibitor chelerythrine increased the activity of channel currents. It is concluded that in rabbit ear artery myocytes there is a constitutively active Ca2+-permeable cation channel that is regulated by external Ca2+ ions and suppressed by tonic PKC activity. It is proposed that this mechanism may contribute to the resting membrane conductance and basal Ca2+ influx in this particular arterial preparation.

Alkaloids↗
PubMed2003

Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression.

The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine-mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (2-4 months) and moderately aged (16-18 months) mice. Post-ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 +/- 2 vs. 18 +/- 2 mmHg in young) and reduced developed (37 +/- 3 vs. 83 +/- 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 +/- 2 vs. 16 +/- 2 IU g-1 in young) whereas the incidence of tachyarrhythmias was similar in young (15 +/- 1 %) and aged hearts (16 +/- 1 %). Functional analysis confirmed equipotent effects of 50 micro M adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 micro M adenosine improved diastolic (5 +/- 1 mmHg) and developed pressures (134 +/- 7 mmHg) and LDH loss (6 +/- 2 IU g-1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to ~10 %). In contrast to adenosine, 50 micro M diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 micro M 5-hydroxydecanoate (5-HD) and 3 micro M chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 micro M 8-sulfophenyltheophylline) and A1-selective (150 nM 8-cyclopentyl-1,3-dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real-time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (~8 fmol mg-1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring adenosine-mediated protection. These data reveal impaired protection via exogenous and endogenous adenosine contributes to ischaemic intolerance with ageing. This is independent of A1AR expression, and involves ineffective activation of a 5-HD-/diazoxide-sensitive process. The effects of A1AR overexpression indicate that the age-related failure in signalling can be overcome.

Adenosine↗
PubMed2004

Effects of angiotensin II on the pericyte-containing microvasculature of the rat retina.

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte-containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated-patch pipettes, measured pericyte calcium levels with fura-2 and visualized pericyte contractions and lumen constrictions by time-lapse photography. We found that angiotensin activates nonspecific cation (NSC) and calcium-activated chloride channels; the opening of these channels induces a depolarization that is sufficient to activate the voltage-dependent calcium channels (VDCCs) expressed in the retinal microvasculature. Associated with these changes in ion channel activity, intracellular calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments revealed that an influx of calcium through the NSC channels is an essential step linking the activation of AT(1) angiotensin receptors with pericyte contraction. Although not required in order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to enhance the contractile response of these cells. In addition to activating nonspecific cation, calcium-activated chloride and voltage-dependent calcium channels, angiotensin II also causes the functional uncoupling of pericytes from their microvascular neighbours. This inhibition of gap junction-mediated intercellular communication suggests a previously unappreciated complexity in the spatiotemporal dynamics of the microvascular response to angiotensin II.

Alkaloids↗
PubMed2006

Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons.

Intracellular Ca2+ regulates a variety of neuronal functions, including neurotransmitter release, protein phosphorylation, gene expression and synaptic plasticity. In a variety of cell types, including neurons, Ca2+ is involved in actin reorganization, resulting in either actin polymerization or depolymerization. Very little, however, is known about the relationship between Ca2+ and the actin cytoskeleton organization in retinal neurons. We studied the effect of high-K+-induced depolarization on F-actin organization in salamander retina and found that Ca2+ influx through voltage-gated L-type channels causes F-actin disruption, as assessed by 53 +/- 5% (n = 23, P < 0.001) reduction in the intensity of staining with Alexa-Fluor488-phalloidin, a compound that permits visualization and quantification of polymerized actin. Calcium-induced F-actin depolymerization was attenuated in the presence of protein kinase C antagonists, chelerythrine or bis-indolylmaleimide hydrochloride (GF 109203X). In addition, phorbol 12-myristate 13-acetate (PMA), but not 4alpha-PMA, mimicked the effect of Ca2+ influx on F-actin. Activation of ionotropic AMPA and NMDA glutamate receptors also caused a reduction in F-actin. No effect on F-actin was exerted by caffeine or thapsigargin, agents that stimulate Ca2+ release from internal stores. In whole-cell recording from a slice preparation, light-evoked 'off' but not 'on' EPSCs in 'on-off' ganglion cells were reduced by 60 +/- 8% (n = 8, P < 0.01) by cytochalasin D. These data suggest that elevation of intracellular Ca2+ during excitatory synaptic activity initiates a cascade for activity-dependent actin remodelling, which in turn may serve as a feedback mechanism to attenuate excitotoxic Ca2+ accumulation induced by synaptic depolarization.

Actins↗