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PubMed2005

Evidence that insulin-like growth factor-1 requires protein kinase C-epsilon, PI3-kinase and mitogen-activated protein kinase pathways to protect human vascular smooth muscle cells from apoptosis.

Insulin-like growth factor (IGF)-1 has been implicated in the development of occlusive vascular lesions. Although its role in vascular smooth muscle cell (VSMC) growth and migration are fairly well characterized, anti-apoptotic signals of IGF-1 in human VSMC remain largely unknown. In this study, we examined IGF-1 signals that protect human and rat VSMC from staurosporine (STAU)- and c-myc- induced apoptosis, respectively. Treatment with STAU resulted in apoptotic DNA fragmentation, phosphatidylserine externalization and cell shrinkage, but only occasional VSMC 'blebbing'. STAU-induced death and IGF-1-mediated survival were concentration dependent, while time-lapse video microscopy showed that IGF-1 inhibited c-myc-induced apoptosis by 90%. Pretreatment with mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitors UO126 and PD098059, or with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, reversed IGF-1-mediated human VSMC survival by 25-27% and 66%, respectively. Translocation studies showed that IGF-1 activated protein kinase C (PKC)-epsilon, but not PKC-alpha or PKC-delta, even in the presence of STAU, while pharmacological PKC inhibition (Ro-318220 or Go6976) implicated PKC-zeta or a novel PKC isozyme in IGF-1-mediated survival. Transient expression of activated PKC-epsilon but not activated PKC-zeta decreased myc-induced apoptosis in rat VSMC. In human VSMC, antisense oligodeoxynucleotides to PKC-epsilon partially reversed IGF-1-induced survival. In addition, IGF-1 elicited a mild but sustained activation of extracellular signal regulated kinase (ERK)1/2 in human VSMC that was abolished after 1 h in the presence of STAU. PKC downregulation reversed both IGF-1- and PMA-induced ERK activity, but platelet-derived growth factor (PDGF)-induced activity was unchanged. These results indicate for the first time that IGF-1 can protect human VSMC via multiple signals, including PKC-epsilon, PI3-K and mitogen-activated protein kinase pathways.

Apoptosis↗
PubMed2001

The effect of N-acetylcysteine on the microscopic fluid dynamics of rat tears.

BACKGROUND: The classical view of the tear film is of a 10-micron film of aqueous tears, sandwiched between thin layers of lipid and mucus. This has been challenged recently by the revelation that the tear film may be considerably thicker than 10 microns and that dissolved mucus and protein may play a much more important role than simply promoting tear adherence. In particular, the primary role of mucus may be to form a structured aqueous gel that adheres closely to the corneal surface and evens out its irregularities, thus providing a high-quality optical surface. METHODS: We have used the robust tear film of the rat eye as an animal model to investigate the contribution of mucus and low-molecular-weight (LMW) proteins to tear film structure. The ocular surface was first exposed to saline, which washed away the tear film. Single drops of a tear/saline mixture, treated with various concentrations of the thiol-reducing agent N-acetylcysteine (NAC), were placed on the ocular surface and the resulting fluid behaviour was recorded with video-microscopy. RESULTS: At five per cent concentration, NAC appeared to degrade the gap-filling and anti-evaporative qualities of the tears, features that give the rat tear film its robust characteristics. Lower concentrations had no significant effect. DISCUSSION: In a previous publication, we showed that five per cent NAC alters the profile of LMW proteins in rat tears. The present observations suggest that the robust wetting properties of rat tears depend critically on their mucus and/or LMW protein content and possibly are related to the formation of an aqueous/mucous gel.

Journal Article↗
PubMed2004

Functional importance of Ca2+-activated K+ channels for lysophosphatidic acid-induced microglial migration.

Abstract Migration of microglial cells towards damaged tissue plays a key role in central nervous system regeneration under pathological conditions. Using time lapse video microscopy we show that lysophosphatidic acid (LPA) enhances chemokinetic migration of murine microglial cells. In the presence of 1 micro m LPA, the mean migration rate of microglial cells was increased 3.8-fold. In patch-clamp studies we demonstrate that LPA induces activation of a Ca(2+)-activated K(+) current. Microglial Ca(2+)-activated K(+) currents were abolished by either 50 nm charybdotoxin or 10 micro m clotrimazole. In contrast, 5 micro m paxilline did not have any significant effects on Ca(2+)-activated K(+) currents. The LPA-stimulated migration of microglial cells was inhibited by blockers of IKCa1 Ca(2+)-activated K(+) channels. The mean migration rate of LPA-stimulated cells was decreased by 61% in the presence of 50 nm charybdotoxin or by 51% during exposure to 10 micro m clotrimazole. Microglial migration was not inhibited by 5 micro m paxilline. It is concluded that IKCa1 Ca(2+)-activated K(+) channels are required for LPA-stimulated migration of microglial cells.

Animals↗
PubMed1999

Mechanisms underlying spontaneous rhythmical contractions in irideal arterioles of the rat.

1. Mechanisms underlying spontaneous rhythmical contractions have been studied in irideal arterioles of the rat using video microscopy and electrophysiology. 2. Rhythmical contractions (4 min-1) were more common during the second and third postnatal weeks and were always preceded by large, slow depolarizations (5-40 mV). 3. Spontaneous contractions were unaffected by tetrodotoxin (1 microM), neurotransmitter receptor antagonists, the sympathetic neurone blocker, guanethidine (5 microM) or sensory neurotoxin, capsaicin (1 microM). 4. Stimulation of sensory nerves inhibited spontaneous activity and this was not prevented by L-NAME (10 microm). 5. L-NAME (10 microm) caused an increase in frequency of spontaneous contractions, while forskolin (30 nM), in the presence of L-NAME, abolished spontaneous, but not nerve-mediated, contractions. 6. Spontaneous activity was not affected by felodipine (1 nM) or nifedipine (1 microM), but was abolished by cadmium chloride (1 microM) or superfusion with calcium-free solution. 7. Caffeine (1 mM), thapsigargin (2 microM) and cyclopiazonic acid (3 microM), but not ryanodine (3 microM), abolished spontaneous and nerve-mediated contractions. After preincubation in L-NAME (10 microM), cyclopiazonic acid abolished spontaneous contractions only. 8. Spontaneous depolarizations and contractions were abolished by 18alpha-glycyrrhetinic acid (20 microM). 9. Results suggest that spontaneous rhythmical contractions are myogenic and result from the cyclical release of calcium from intracellular stores, without a contribution from voltage-dependent calcium channels. Intercellular coupling through gap junctions appears to be essential for co-ordination of these events which could be modulated by nitric oxide and increases in cAMP. The possibility that different intracellular stores underly spontaneous and nerve-mediated contractions is discussed.

Adenosine Triphosphate↗
PubMed1994

Vapor phase exposure to acetaldehyde generated from ethanol inhibits bovine bronchial epithelial cell ciliary motility.

Acetaldehyde is a toxic product of the oxidation of ethanol and is known to induce slowing of ciliary motility in airway epithelium. Alcohol ingestion results in high exhaled breath concentrations of ethanol where lung microsomes and upper airway bacterial flora are capable of metabolizing it to acetaldehyde. Because acetaldehyde is very volatile, we hypothesized that the production and release of acetaldehyde vapor into the airway may result in ciliary slowing or ciliastasis. To test this hypothesis, ciliated bovine bronchial airway epithelial cells were maintained on collagen-coated dishes for 48 hr before coincubation with a separate dish containing control or test mixtures. In this arrangement, the ciliated cells were exposed only to the volatile components of the separate dish. The separate dish contained ethanol, acetaldehyde, or an acetaldehyde-generating system (AGS) that consisted of ethanol + glucose + glucose oxidase + catalase. This mixture was placed next to the ciliated cells within in a transparent sealed chamber. Ciliary motility was recorded at room temperature by video microscopy, and ciliary beat frequency was determined using computerized frequency spectrum analysis. Exposure to the AGS resulted in time- and concentration-dependent ciliary slowing with complete ciliastasis, with as low as 20 mM ethanol in the AGS. Direct vapor phase exposure to acetaldehyde alone served as a positive control and also resulted in time-dependent ciliary slowing with complete ciliastasis reached by 4 hr. The AGS-induced ciliastasis was reached 2 hr later than with acetaldehyde alone. When cells were pretreated with cyanamide, which is known to block acetaldehyde dehydrogenase, the time to ciliastasis was decreased by 10-30 min compared with untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMedDate not supplied

Trichomonas vaginalis: ultrastructural bases of the cytopathic effect.

The in vitro cytopathic effect of Trichomonas vaginalis on epithelial cells was explored through the interaction of trophozoites of the virulent strain GT-10 with MDCK monolayers. The interaction was analyzed through electrophysiology, video microscopy, and transmission and scanning electron microscopy. Electrical measurements revealed that living parasites produced severe damage to the cell monolayers within 30 min, manifested as a rapid decrease in transepithelial resistance. Microscopic observations demonstrated that when placed in contact with epithelial cells, trichomonas formed clumps through interdigitations and transient plasma membrane junctions between adjacent parasites. Also, attached trophozoites adopted an ameboid shape. The in vitro cytopathic action of T. vaginalis on MDCK cells was initially evident by modifications of the plasma membrane, resulting in opening of tight junctions, membrane blebbing, and monolayer disruption. After 15 min of interaction the damage was focal, concentrating at sites where parasite clumps adhered to the monolayer. At 30 min practically all MDCK cells were dead, whether or not trichomonas were attached to them. These events were followed by detachment of lysed cells and complete disruption of the monolayer at 60 min. Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T. vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized. The same sequence of lytic events was found with the less virulent GT-3 strain. However, the time course of cytolysis with GT-3 parasites was much slower, and lysis was limited to areas of attachment of T. vaginalis.

Adult↗
PubMedDate not supplied

Contributions of the axostyle and flagella to closed mitosis in the protists Tritrichomonas foetus and Trichomonas vaginalis.

Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonad's cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

Animals↗
PubMedDate not supplied

Activated c-Met signals through PI3K with dramatic effects on cytoskeletal functions in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive illness with early metastases. There are several receptor tyrosine kinases (RTKs) overexpressed in SCLC, including c-Met. c-Met contains an external semaphorin-like domain, a cytoplasmic juxtamembrane domain, tyrosine kinase domain and multiple tyrosines that bind to adapter molecules. We have previously reported that c-Met is abundantly expressed in the NCI-H69 SCLC cell line and now have determined the downstream effects of stimulating c-Met via its ligand hepatocyte growth factor (HGF). Utilizing unique phospho-specific antibodies generated against various tyrosines of c-Met, we show that Y1003 (binding site for c-Cbl and a negative regulatory site), Y1313 (binding site for PI3K), Y1230/Y1234/Y1235 (autophosphorylation site), Y1349 (binding site for Grb2), Y1365 (important in cell morphogenesis) are phosphorylated in response to HGF (40 ng/ml, 7.5 min) in H69 cells. Since multiple biological and biochemical effects are transduced through the PI3K pathway, we determine the role of PI3K in the c-Met/HGF stimulation pathway. We initially determined that by inhibiting PI3K with LY294002 (50 microM over 72 hours), there was at least a 55% decrease in viability of H69 cells. Since H69 SCLC cells form clusters in cell culture, we determined the effects of HGF and LY294002 on cell motility of the clusters by time-lapse video microscopy. In response to HGF, SCLC moved much faster and formed more clusters, and this was inhibited by LY294002. Finally, we determined the downstream signal transduction of HGF stimulation of c-Met with and without inhibition of c-Met (with geldanamycin, an anisamycin antibiotic that inhibits c-Met in SCLC) or PI3K (with LY294002). We show that association of c-Met with PI3K and GAB2 is diminished by inhibiting c-Met. In summary, activation of the c-Met pathway targets the PI3K pathway in SCLC and this may be an important therapeutic target.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Basal cell carcinoma--an in-vivo model of human tumour microcirculation?

The role of angiogenesis in tumour growth and metastasis is well established. However, investigations of tumour microcirculation to date have used either biopsy material from human tumours, or animal models in vivo. We have studied the tumour microcirculation in vivo in human skin cancers using video-microscopy to examine 12 basal cell carcinomas (BCCs) on the head and neck of 11 patients, and compared the vessels with those seen in the peri-lesional skin, and in normal control skin on the opposite side of the body. The vessels seen within the BCCs were markedly abnormal qualitatively, forming bizarre, disorganized patterns. Quantitative analysis revealed that the mean diameter (+/-standard deviation) of the largest vessel was significantly greater within the BCC (0.086+/-0.029 mm) than that in the control skin (0.034+/-0.012 mm) (P<0.001). The area fraction, a measure of the area of tissue occupied by vessels, was increased highly significantly within the BCCs (0.158+/-0.038) compared with both peri-lesional skin (0.029+/-0.012) and control skin (0.027+/-0.010) (P<0.001). Length density, the length of blood vessel per unit area of tissue, was also highly significantly greater within the lesion (210.22+/-66.05 cm(-1)) compared to peri-lesional (27.10+/-15.67 cm(-1)) and control skin (28.27+/-15.81 cm(-1)) (P<0.001). This is the first study to have demonstrated that BCCs possess a distinct tumour microcirculation which can be observed directly, and assessed quantitatively. Prospective studies of tumour progression, possibly after intervention with angiogenesis inhibitors, are possible.

Aged↗
PubMed1993

The cytosolic chloride concentration in macula densa and cortical thick ascending limb cells.

It is believed that chloride transport through the macula densa (MD) cells is a factor involved in the tubuloglomerular feedback (TGF) mechanism and in MD-mediated renin release. In this study isolated and perfused rabbit kidney cortical thick ascending limb (cTAL) segments containing MD plaques and attached glomeruli were loaded with chloride (CL-sensitive) 6 methoxy-1-fluorophore (sulphanate-propyl) quinolinium (SPQ). MD and cTAL intracellular chloride concentration ([Cl-]i) was determined by using image-intensified video microscopy and digital image-processing for measuring the intensity of the emitted SPQ fluorescence. With 150 mM NaCl in lumen and bath the [Cl-]i in MD and cTAL cells was 58.8 +/- 7.2 mM (n = 20) and 68.7 +/- 9.8 mM (n = 14), respectively. When the presumed luminal Na(+)-2Cl(-)-K+ co-transporter was blocked by adding 10(-4)M furosemide, the [Cl-]i was reduced in both, MD and cTAL cells from 55.5 +/- 11.9 to 28.6 +/- 10.0 mM (n = 10) and from 43.8 +/- 2.6 to 13.1 +/- 4.5 mM (n = 5), respectively. A reduction in luminal NaCl from 150 to 30 mM also decreased both, MD and cTAL [Cl-]i from 69.4 +/- 9.1 to 36.5 +/- 5.1 mM (n = 9) and from 82.9 +/- 14.5 to 49.4 +/- 8.0 mM (n = 8), respectively. Basolateral addition of the Cl(-)-channel blocker NPPB increased MD [Cl-]i from 31.1 +/- 2.0 to 100.7 +/- 17.0 mM (n = 5) and cTAL [Cl-]i from 44.4 +/- 12.9 to 89.7 +/- 11.7 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Photosensory transduction in ciliates. I. An analysis of light-induced electrical and motile responses in Stentor coeruleus.

Light-induced membrane potential changes and motile responses have been studied in Stentor cells with intracellular microelectrodes and video microscopy, respectively. Intracellular microelectrode recordings showed that step-up increase in light intensity induced an electrical membrane response which consisted of an initial membrane depolarization (photoreceptor potential) followed by an action potential and maintaining phase of depolarization (afterdepolarization). The amplitude of the receptor potential is dependent on the intensity of light stimulus and the action potential appears with a lag period (latency) after the onset of light stimulus. The extent of the membrane afterdepolarization is dependent on the intensity and duration of stimulus used. A close time correlation has been established between the latency for the action potential and the onset of ciliary reversal (stop response). A time correlation was also observed between the duration of the membrane afterdepolarization and the duration of backward swimming. The action spectrum for the photoreceptor potential amplitude of Stentor resembled the action spectra for the latency of ciliary reversal and the photoresponsiveness, indicating that the photomovement response and membrane potential changes are coupled through the same photosensor system. A hypothesis on the photosensory transduction chain in Stentor is discussed according to which the photoreceptors and the ciliary apparatus is mediated by the membrane potential changes.

Animals↗
PubMed1995

Influence of stretch on excitation threshold of single frog ventricular cells.

The excitation threshold of enzymatically dissociated single frog ventricular myocytes was measured as a function of cell length or sarcomere length. Field stimulation with 3 ms duration rectangular current pulses was applied via two platinum wire electrodes oriented either parallel or perpendicular to the long axis of the cell. Excitation threshold was measured as the amplitude of applied current, with an error of less than 1%. Single cells were held isometrically by two glass pipettes and wrapped around an optical fibre, which was used to control cell length. Sarcomere length was measured using video microscopy and a phase-locked loop system. Changes in cell length and sarcomere length were induced by mechanical stretch and release of the whole cell. The excitation threshold decreased with increasing resting length (preload), and increased with decrease of the cell length back to its original length, regardless of the orientation of the electric field. The measured change in excitation threshold with stretch was of the order of 1%/% strain. These results suggest that stretch affects the excitation threshold of cardiac cells by factors other than simple shape changes in the cell, perhaps through the activity of mechanosensitive ionic channels.

Action Potentials↗
PubMed1976

Intercellular coupling in the atrioventricular node and other tissues of the rabbit heart.

1. A fluorescent tracer dye, sodium fluorescein (mol.wt. 332), was used to assess the relative degree of intercellular coupling in various tissues of the rabbit heart. 2. Dye was injected intracellularly by micro-iontophoresis. Subsequent movement into contiguous cells was monitored by video microscopy. From these data the permeability of the intercellular boundaries was computed. 3. The values of boundary permeability were consistent with those expected from previous studies with tracers whose molecular weights bracketed that of fluorescein. 4. In the atrium, ventricle, Purkinje strands and His bundle, the relative magnitude of the boundary permeability correlated reasonably well with the relative profusity of gap junctional area on the intercalated disk, the latter estimated from published data. 5. The rate of passage of dye between N cells of the atrioventricular, AV, node was at least three orders of magnitude lower than between cells of the other tissues studied; this result is consistent with published reports indicating few gap junctions between cells within the region of slow conduction. 6. Quantitative considerations based on these data indicate that N cells may not be sufficiently well coupled to permit impulse propagation through the AV node by intercellular current flow, alone.

Animals↗
PubMed1995

Flow-dependent regulation of arteriolar diameter in rat skeletal muscle in situ: role of endothelium-derived relaxing factor and prostanoids.

1. Arteriolar diameter in the resting rat spinotrapezius muscle was studied by intravital video microscopy before and after blockade of the L-arginine-EDRF (NG-nitro-L-arginine, L-NNA) or the cyclo-oxygenase-prostacyclin (indomethacin) pathway. Blockade of either pathway leads to a decrease of arteriolar diameter of 25-40%, while the combined blockade of both results in vasoconstriction of 50-60%. 2. Alteration of blood flow velocity elicited by partial micropipette occlusion induces corresponding changes of vessel diameter. The flow-dependent diameter response is reduced by about 80% by L-NNA. By contrast, blockade of prostanoid production shows no significant influence on vessel response to blood flow alteration in the range tested. 3. Transient overshooting vasodilatation is seen for about 1 min following the sudden restoration of flow velocity subsequent to occlusion. In contrast to the initial phase of this response, the late phase is blocked by L-NNA. 4. The findings suggest that basal release of endothelium-derived relaxing factor (EDRF) and prostanoids leads to additive and independent dilator effects, and that flow-dependent diameter changes are primarily mediated by EDRF. 5. If present data are compared with literature reports, it appears that arterial flow sensitivity is most pronounced in the smallest vessels. In such vessels, flow-dependent dilatation will amplify even small changes of volume flow by more than four times.

Animals↗
PubMed1996

Effect of metabolic inhibition on K+ channels in pyramidal cells of the hippocampal CA1 region in rat brain slices.

1. The effect of metabolic inhibition on membrane potential and ionic conductances of K+ channels was studied with the patch-clamp technique in pyramidal cells in the CA1 region of the hippocampus. Individual cells were visualized in brain slices from rats aged between 9 and 19 days using infrared video microscopy. Excitability was inhibited by tetrodotoxin. 2. Dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and cyanide hyperpolarized the majority of the cells. The resting potential (V) was -55.3 +/- 0.23 mV (n = 147). In response to DNP the change in V was -3.9 +/- 0.76 mV (n = 59), with a normal distribution ranging between +9.0 and -16 mV. 3. Metabolic inhibition increased the resting conductance (grest) and the conductance related to the delayed outward current measured at V = -20 mV (g-20), and decreased the conductance of the early outward A-current (gA). The changes in grest and g-20 were transient and differed from the time-dependent changes seen in control cells. 4. Tolbutamide reversed the hyperpolarization and the increase in grest. Glibenclamide, apamin and charybdotoxin were ineffective. 5. The presence of ATP (2 mM) in the pipette solution did not influence the change in grest. ATP did, however, affect the time-dependent decline in gA and g-20, which demonstrated that cells had been perfused. 6. Cadmium (0.5 mM) reduced the increase in g-20 and grest obtained with DNP, although it did not prevent the effect of DNP on grest. This indicates that the action of DNP involves an elevation of intracellular [Ca2+]. 7. It is concluded that metabolic inhibition causes changes in the function of several types of K+ channels in CA1 cells. A transient opening of a tolbutamide-sensitive K+ channel could explain the increase in grest and the hyperpolarization observed in most cells.

Animals↗
PubMed1997

Osmolarity modulates K+ channel function on rat hippocampal interneurons but not CA1 pyramidal neurons.

1. Whole-cell and single-channel recording methods were used in conjunction with infrared video microscopy techniques to examine the properties of voltage-activated potassium channels in hippocampal neurons during the application of hyposmolar solutions to hippocampal slices from rats. 2. Hyposmolar external solutions (osmolarity reduced by 10% to 267 mosmol l-1) produced a significant potentiation of voltage-activated K+ current on lacunosum/moleculare (L/M) hippocampal interneurons, but not on CA1 and subiculum pyramidal neurons. Hyperpolarization-activated (IH) and leak currents were not altered during the application of hyposmolar solutions in all cell types. 3. Mean channel open time and the probability of channel opening were dramatically increased under hyposmolar recording conditions for outside-out patches from L/M interneurons; no changes were observed for patches from CA1 pyramidal neurons. Mean current amplitude and the threshold for channel activation were not affected by hyposmotic challenge. 4. Hyposmolar external solutions produced a significant reduction in the firing frequency of L/M interneurons recorded in current-clamp mode. Hyposmolar solutions had no effect on resting membrane potential, action potential amplitude or duration, and spike after-hyperpolarization amplitude. 5. These results indicate that selective modulation of interneuron ion channel activity may be a critical mechanism by which osmolarity can regulate excitability in the central nervous system.

Animals↗
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

Neuropeptide Y inhibits axonal transport of particles in neurites of cultured adult mouse dorsal root ganglion cells.

Neuropeptide Y (NPY) plays a modulatory role in processing nociceptive information. The present study investigated the effects of NPY on axonal transport of particles in neurites of cultured adult dorsal root ganglion (DRG) cells using video-enhanced microscopy. Application of NPY decreased the number of particles transported in both the anterograde and retrograde directions. This effect was persistently observed during NPY application and was reversed after washout. The inhibitory effect of NPY was concentration dependent between 10(-9) M and 10(-6) M. The instantaneous velocity of individual particles moving in anterograde and retrograde directions was also reduced by NPY. Both the NPY Y1 receptor agonist [Leu31,Pro34]-NPY and NPY Y2 receptor agonist NPY(13-36) mimicked the effect of NPY on the number of transported particles. An immunocytochemical study using an antiserum against the NPY Y1 receptor protein revealed that the Y1 receptor was expressed in the majority (85.9 %) of cultured adult mouse DRG cells. Pre-treatment of cells with pertussis toxin, a GTP-binding protein (G protein) inhibitor, completely blocked the inhibitory effect of NPY. Each application of SQ-22536, an adenylate cyclase inhibitor, and H-89, a protein kinase A inhibitor, mimicked and occluded the effect of NPY. In contrast, dibutyryl cAMP (dbcAMP), a membrane permeable cAMP analogue, and forskolin, an activator of adenylate cyclase, produced a transient increase in axonal transport. The application of dbcAMP and forskolin in combination with NPY negated the effect of NPY alone. These results suggest that NPY, acting at Y1 and Y2 receptors, inhibits axonal transport of particles in sensory neurones. The effect seems to be mediated by a pertussis toxin-sensitive G protein, adenylate cyclase, and protein kinase A pathway. Therefore, NPY may be a modulatory factor for axonal transport in sensory neurones.

Age Factors↗