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PubMed1995

Agreement among evaluators of bovine embryos produced in vivo or in vitro.

Six experienced individuals evaluated 40 embryos on videotape for stage of development and quality grade. These 40 observations comprised 15 embryos produced in vivo, 15 embryos produced in vitro, and 10 embryos that were repeated throughout the videotape. Embryos produced in vivo were recovered from uterine flushings of superovulated heifers 7 d after estrus, and embryos produced in vitro were harvested 7 d after insemination of in vitro-matured oocytes. Embryos of various stages (morulae, blastocysts, or degenerated) and quality grades (1 = excellent, 2 = good, 3 = fair, 4 = degenerated) were recorded on videotape for evaluation. After video microscopy, the embryos were stained and the number of nuclei per embryo was counted. Six evaluators reviewed the videotape and the percentage of agreement and kappa (k; agreement beyond chance) among evaluators were determined for classifications of stage and grade. Consistency of each evaluator's responses was estimated using the 10 repeated embryos. Agreement within evaluators was higher for stage of embryo development (89.2%) than quality grade (68.5%). Agreement among evaluators for stage was slightly higher with embryos produced in vivo (85.0%, k = 0.74) than in vitro (72.3%, k = 0.48). Agreement among evaluators for grade was similar with embryos from in vivo (61.0%, k = 0.46) and in vitro (57.7%, k = 0.42) production. For both sources of embryos, agreement was substantially better for Grades 1 and 4 than for Grades 2 and 3. The results of this study suggest that good to excellent agreement exists for classifying Day 7 bovine embryos by stage and by extremes of quality grade (Grades 1 and 4) but not by degree of abnormal morphology (Grades 2 and 3). Simple grading criteria of Grade 1 (highest quality), Grade 2 (morphologic defects), and Grade 3 (degenerated) maximized agreement among evaluators.

Journal Article↗
PubMed1986

Visualization and analysis of mural thrombogenesis on collagen, polyurethane and nylon.

Visualization and analysis of mural thrombogenesis on collagen-coated glass, polyurethane and nylon was discussed. Epi-fluorescent video microscopy was used to visualize thrombotic events at a protein or polymer surface in contact with flowing whole blood. Digital image processing was used to analyse the real-time microscopic images obtained, resulting in measurements of morphological features and the number of platelets that compose each thrombus. The three-dimensional structures of the thrombi were also estimated. A photodiode was used to measure integrated end-point platelet accumulation at different axial positions along the surface. Additionally, the convection-diffusion equations were solved to estimate the concentrations of adenosine diphosphate, thromboxane A2, and thrombin generated by activated platelets at the blood-contacting surface.

Adenosine Diphosphate↗
PubMed1993

Air-water interface displaces adsorbed bacteria.

Video microscopy was employed to observed the spatial distribution of Staphylococcus epidermidis and Pseudomonas aeruginosa adherent to glass and polymer substrates. During rinsing procedures the bacteria remained in their original positions when the surfaces were rinsed with saline for 3 min followed by ethanol for 3 min before exposure to air. When the surfaces were rinsed with saline only, the air-liquid interface disrupted the spatial distribution of the bacteria, removing and redepositing the bacteria in clumps. A moving air-liquid interface of a gas bubble on substrate also displaced bacteria. Such artefacts produced by air-water interfaces should be avoided during bacterial adhesion experiments.

Adsorption↗
PubMed1996

Kinetics of bone cell organization and mineralization on materials with patterned surface chemistry.

Materials with spatially resolved chemistries (i.e. patterned surfaces) have been used to guide and organize the position of mammalian cells in vitro. A common theme in guiding the spatial distribution of cells has been the use of patterned alkylsiloxanes, where one region contains an aminosilane and the other an alkylsilane. The regions of the aminosilane served as preferential sites for cell attachment and spreading, presumably dependent on the association between cell surface proteoglycans the positively charged amine. In this study, experiments were conducted with patterns of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS) to determine the kinetics of spatial organization of bone-derived cells, and whether initial attachment and spreading affected the rate of matrix mineralization (i.e. bone formation) in extended cultures. The bone cells required the presence of serum or preadsorption of serum proteins to the patterned EDS/DMS surface to organize according to the lithographically defined surface chemistry. Time-lapse video microscopy indicated that cells were randomly distributed over the EDS/DMS surface at the time of plating, but organized on the EDS regions within 30 min. When cultures were extended for 15 and 25 days, the matrix synthesized by the cells was preferentially mineralized on the EDS chemistry. These results demonstrate the ability of surface chemistry modifications to organize cells and form mineralized tissue in vitro. The methods employed should have general value to the engineering of tissues in vitro.

Animals↗
PubMed1988

Calcium dependence of bleb formation and cell death in hepatocytes.

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate ('chemical hypoxia'). Cytosolic free Ca2+ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca2+ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca2+ increased rapidly in high Ca2+ buffer (1.2 mM) but not in low Ca2+ buffer (less than 1 microM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca2+ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca2+ buffer, cytosolic free Ca2+ increased in parallel with dye leakage. No regional changes in cytosolic free Ca2+ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca2+ was not required for the metastable state to develop or cell death to occur.

Aged↗
PubMed1993

A discrete Na+/Ca2+ exchange dependent, Ca2+ compartment in cultured neonatal rat heart cells. Characteristics, localization and possible physiological function.

A pool of calcium has been identified in cultured neonatal rat heart cells whose exchange is dependent on extracellular sodium and calcium. In the absence of extracellular sodium and calcium this pool retains at least 760 mumol calcium/kg dry weight, which is released upon the introduction of sodium and calcium, with a t1/2 of 4.4 s. Video microscopy experiments of non-stimulated cells showed that the cells, despite the retention of 760 mumoles calcium/kg dry weight, do not develop contracture, indicating that the majority of the pool is not retained in the general cytoplasm. Neither is the pool displaced by lanthanum. Several probes for the sarcoplasmic reticulum were used. Ryanodine (1 microM) had no significant effect on the size of the pool. Caffeine reduced the size of the pool by 60% and thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca-ATPase reduced the content of the pool by 70%. High concentrations of ryanodine (1 mM) reduced the pool even further. The experimental data, in association with recently developed concepts of the diadic region [1], indicate that the pool may reside in the sub-sarcolemmal space between the sarcoplasmic reticulum and the sub-sarcolemma. The physiological significance of this pool is discussed.

Animals↗
PubMed1996

Neutrophil adhesion on surfaces preadsorbed with high molecular weight kininogen under well-defined flow conditions.

The adhesion of neutrophils and other leukocytes to biomaterial surfaces is an important phenomenon in the host response to biomaterials because the number of adherent leukocytes is often related to the inflammatory response after implantation. After adhering to biomaterial surfaces, other leukocyte reactions, such as phagocytosis, respiratory burst, and protease release, may occur and result in the deterioration of the implanted biomaterial and injury to peripheral tissue. This study of neutrophil adhesion quantitatively characterizes neutrophil adhesion under well-defined laminar flow conditions using a radial flow chamber. In this rheologically well-defined system, the fluid shear rate on the surface varies continuously with radial position. This allows the study of shear-dependent behavior of neutrophil adhesion. Exploiting the variable shear rate in the radial flow chamber, the kinetics of neutrophil adhesion was obtained using automated video microscopy and image analysis to recursively acquire cell counts from multiple fields in different radial positions, and to quantify the surface density of neutrophil as a function of time. Neutrophil adhesion was studied on glass preadsorbed with fibrinogen and high-molecular-weight kininogen (HK). At a shear rate of 20 s-1, the number of adherent cells on the preadsorbed fibrinogen surface was similar to that on bare glass, and the number of adherent cells on the HK surface was less than 10% of that on the bare glass. We conclude that surfaces preadsorbed with HK are anti-adhesive to neutrophils.

Adsorption↗
PubMed1992

N-methyl-D-aspartate effects on the growth, morphology and cytoskeleton of individual neurons in vitro.

Short-term (up to 5 h post-plating) cerebellar granule cell cultures were prepared from the week-old rat and maintained in a micro-incubator during time lapse video microscopy to examine normal and N-methyl-D-aspartate (NMDA)-evoked neurite extension. In untreated cultures growth of neurites was stochastic but proceeded at an average rate of 12.0 +/- 1.4 microns/h. Growth cone morphology was variable. The classical filopodia and lamellipodia possessing tips were motile or non-motile, while those processes ending in a club shape were rarely seen to extend. Individual growth cones passed through several different morphologies during growth. New processes extended more rapidly (13.0 +/- 1.7 microns/h) than those already present (9.0 +/- 0.5 microns/h). Addition of the NMDA receptor antagonist, aminophosphonovalerate (APV), caused a marked retraction of pre-existing processes. Stimulation of the receptor with 50 microM NMDA caused a marked increase in growth rate compared to controls (15.0 microns/h and 1.7 microns/h, respectively). When the presence of actin-rich structures was examined using rhodamine-phalloidin labelling it was found that NMDA increased the proportion of neuronal processes that possessed a growth cone by 28%. Conversely, inhibition of NMDA receptor activity with APV reduced the formation of lamellipodia from neuronal cell bodies.

2-Amino-5-phosphonovalerate↗
PubMed1994

Selective constriction of small cutaneous arteries by NPY matches distribution of NPY in sympathetic axons.

This study has begun to investigate some functional implications of the differential localization of neuropeptide Y (NPY) in sympathetic neurons supplying different arterial segments in the cutaneous circulation of the guinea-pig ear. Responses of the main ear artery to exogenous NPY and norepinephrine (NE) were examined in vitro by measuring isometric tension. Responses of smaller arterial vessels to application of exogenous NPY or NE to the adventitial surface were examined in anaesthetized, ventilated guinea-pigs, by measuring changes in internal vessel diameter using video microscopy. Some arterial segments subsequently were examined for the presence of immunoreactivity (IR) to tyrosine hydroxylase (TH) and NPY. NPY (1 nM-10 microM) contracted the main ear artery (EC50 = 10 nM; max. contraction = 30% KCl), and 1 nM NPY produced slight potentiation of contractions produced by NE. In vivo, local applications of NPY (1-10 microM) constricted only a subpopulation of arterial vessels (23 of 41). All vessels constricted by NPY were innervated by axons containing IR to both TH and NPY, and as a population, were more proximal in the arterial tree (branch orders 3 to 6) than were vessels insensitive to NPY (branch orders 4 to 8). Most vessels insensitive to NPY were arterioles and arterio-venous anastomoses < 40 microns in diameter, which were innervated by axons containing TH-IR but not NPY-IR. In contrast, local application of NE (1-30 microM) constricted all vessels examined in vivo. When present, NPY constrictions had a longer latency (15-45 s) and duration (3-4 min) than NE constrictions of the same vessel segments. In vivo, NPY sometimes potentiated the peak amplitude of NE constrictions (2 of 7 vessels), but only in vessels where NPY also produced direct constriction. These results reveal an excellent correlation between the localization of NPY in sympathetic axons, and the location of postsynaptic NPY receptors throughout the cutaneous arterial system. Any NPY released in response to strong activation of cutaneous sympathetic neurons is likely to act preferentially on the proximal cutaneous arteries, and to lead to a more prolonged constriction of these arteries than of more distal arterioles and arterio-venous anastomoses.

Adrenergic Fibers↗
PubMed1992

Inhibition of platelet adhesion and thrombus formation on a collagen-coated surface by novel carbamoylpiperidine antiplatelet agents.

The inhibitory effect of two novel antiplatelet agents, 1,10-bis[3-(N,N-diethylcarbamoyl)piperidino]decane dihydrobromide (G-110) and 1,6-bis[3-(N,N-diethylcarbamoyl)piperidino]hexane dihydrobromide (G-112), on platelet adhesion and subsequent aggregation on collagen-coated surface was evaluated under controlled flow. Glass coverslips coated with bovine fibrillar collagen type I were exposed to heparinized human whole blood that had been preincubated with either aqueous solutions of one of the two carbamoylpiperidine congeners or corresponding amounts (1.0-4.0 microliters/ml blood) of distilled water, at a wall shear rate of 1000 s-1, in a parallel-plate perfusion chamber. Epifluorescence video microscopy with a microphotometric measurement technique was used to visualize and quantify deposition of fluorescently-labeled platelets from flowing whole blood onto the collagen-coated surface. At concentrations of 100 and 200 microM, G-110 inhibited platelet accumulation by 30 +/- 9% (+/- S.E.) and 63 +/- 3% (+/- S.E.), respectively; while G-112 reduced platelet deposition by 19 +/- 3% (+/- S.E.) and 31 +/- 2% (+/- S.E.) at concentrations of 200 and 400 microM, respectively. Digital image processing techniques were used to analyze the dynamics of thrombus growth on the collagen-coated surfaces. It was found that the compounds reduced the rate of thrombus growth by impeding both surface coverage and the number of platelets per thrombus in a concentration-dependent manner. This study, together with others on related compounds GT-12, BPAT-117 and BPAT-143, corroborates the nature of pivotal features in the molecular structure of carbamoylpiperidine and nipecotoylpiperazine derivatives which enhance desirable antithrombotic effects, e.g. intramolecular distance between two tertiary amines (ring nitrogens) and levels of hydrophobicity.

Animals↗
PubMed1991

Efflux of protons from acidic vesicles contributes to cytosolic acidification of hepatocytes during ATP depletion.

The objective of this study was to determine the relationship between cytosolic pH and vesicular pH during ATP depletion. Using digitized video microscopy and single, cultured rat hepatocytes, cytosolic pH and vesicular pH were quantitated by ratio imaging of BCECF (2', 7' biscarboxyethyl-5,6-carboxyfluorescein) fluorescence and fluorescein-dextran fluorescence, respectively. Basal value for cytosolic pH was 7.26 and basal value for vesicular pH was 4.86. During ATP depletion by metabolic inhibition with KCN plus iodoacetic acid or antimycin A, cytosolic pH decreased 0.71 units to 6.55. In separate experiments under identical conditions, vesicular pH increased 1.59 units to 6.45, suggesting that protons were leaking from acidic vesicles during ATP depletion. Fluorescein-dextran fluorescence remained punctate, indicating that the rise in vesicular pH was due to an efflux of protons from vesicles and not loss of vesicle integrity. To determine whether efflux of protons from acidic vesicles can acidify cytosolic pH, we used two maneuvers that result in leakage of protons from acidic vesicles without significantly decreasing cellular ATP: (a) hypotonic stress in K(+)-free media and (b) exposure of the cells to the H(+)-ATPase inhibitor NBD-Cl. Both hypotonic stress and NBD-Cl decreased cytosolic pH 0.4 units to 6.86 and increased vesicular pH 2.0 units to 6.76, resulting in near-equilibration of cytosolic pH and vesicular pH. Thus an efflux of protons from intracellular compartments will acidify cytosolic pH of hepatocytes (pH 6.86), but not to the same degree as ATP depletion (pH 6.55).(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗
PubMed1994

Mitochondrial dysfunction during anoxia/reoxygenation injury of liver sinusoidal endothelial cells.

Sinusoidal endothelial cell injury plays a pivotal role in anoxia/reoxygenation liver damage. However, the mechanisms culminating in anoxia/reoxygenation endothelial cell injury remain unclear. Our aims were to determine whether anoxia/reoxygenation injury of sinusoidal endothelial cells causes mitochondrial dysfunction. In cultured rat liver sinusoidal endothelial cells, the mitochondrial membrane potential, cytosolic free calcium and cytosolic pH were quantitated by means of fluorescent probes and multiparameter digitized video microscopy. Cell viability was measured on the basis of lactate dehydrogenase release, and ATP was quantitated with a luciferin/luciferase assay. Mitochondrial membrane potential was stable during 90 min of aerobic perfusion. After 60 and 90 min of anoxia, mitochondrial membrane potential decreased gradually to 97% +/- 6% and 79% +/- 7% of the basal value, respectively. However, mitochondrial membrane potential decreased abruptly with reoxygenation after 60 min of anoxia to 45% +/- 12% of the basal value and did not recover over 30 min of aerobic perifusion. Loss of mitochondrial membrane potential could not be attributed to changes of cytosolic free calcium, cytosolic pH, nitric oxide generation or activity of poly(ADP-ribose) polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

The aminoglycoside G418 suppresses muscarinic receptor-activated calcium release in stably transfected murine N1E-115 neuroblastoma cells.

The aminoglycoside G418 inhibited the release of calcium (Ca2+) from internal stores coupled to muscarinic receptors in murine N1E-115 neuroblastoma cells carrying the aminoglycoside resistance gene neomycin phosphotransferase (NPT). No significant effect was observed on responses coupled to histamine or bradykinin receptors. Cells were transfected using the eukaryotic expression vector pH beta APr-1-neo and selected using G418. Two groups were differentiated either in the continued presence of G418 or in the absence of G418. Carbachol (1 mM), histamine (200 microM) and bradykinin (100 nM) were administered to cells for thirty seconds and changes in [Ca2+]i were measured with fluorescence video microscopy of single cells loaded with the Ca2+ indicator fura-2. The effects of G418 on carbachol evoked Ca2+ release included a 73% reduction in the number of cells responding, a two fold increase in the time to reach half-maximal response, a 35% reduction of the peak [Ca2+]i in response to agonist and an elevation of resting [Ca2+]i from 99 +/- 14 nM (mean +/- S.E.M.) to 155 +/- 27 nM. Acute application (20 min) of G418 to transfected cells differentiated without G418 also reduced the percentage of cells responding to carbachol. This effect was less pronounced in non-transfected parent cells. Thus, the mechanism might involve a metabolite of G418 produced in cells expressing NPT. These results indicate that G418 attenuates Ca2+ release coupled to muscarinic receptors.

Animals↗
PubMed1994

Shortening of a severed squid giant axon is non-uniform and occurs in two phases.

The shortening of severed squid giant axons (GAs) in vitro was analyzed using video light microscopy. Axonal shortening occurred in two temporal phases along the length of the GA: a rapid initial phase during the first 3.5 min after severance followed by a slower phase lasting at least 30 min. The rate of shortening was greatest near the cut end and declined with distance from the cut end for at least 30 min after transection. Axonal shortening may help pack injury-induced vesicles [3] which facilitate sealing of the cut end [7] and/or retard the entry of various substances.

Actins↗
PubMed1991

Skin suction blister wound exposed to u.v. irradiation: a burn wound model for use in humans.

The effects of exposing blister wounds to u.v. irradiation were assessed in 14 male volunteers, on whose forearms blister wounds (diameter 5 mm, suction set at 200 mmHg below atmospheric for 2 h 15 min, blister roof cut at base), and irradiated blister wounds (as above, in addition u.v. irradiation given selectively for 30 min from a distance of 10 cm), were produced. In non-u.v.-irradiated wounds, flow cessation, assessed by video microscopy (n = 6), was observed in a small proportion of the papillary loop vessels. The oedema adjacent to the wound was poorly developed. Laser Doppler linear scans (n = 8) demonstrated a pronounced hyperaemia in the wound bed and also in the adjacent skin, the reaction subsiding over a few days. The exudation rate, determined by weighing the hydrocolloid dressings applied to the wound, was maximal on day 1 and then rapidly decreased. Epithelialization, assessed evaporimetrically as the time taken for reinstatement of the epidermal water barrier, was complete in 5.1 days. In the u.v.-irradiated wounds the blood flow had ceased in all the papillary loop vessels by day 1, and increased oedema, exudation and hyperaemia at the wound edges were observed. Epithelialization was not significantly retarded by the irradiation injury. After 6 months, slight discolouration of the skin was occasionally observed, but no cosmetically disturbing scars. This benign and standardized wound model in humans--based on a combination of a mechanical suction injury and a superficial radiation burn--may prove to be useful, for instance when studying the effects of burns treatment.

Adult↗
PubMed1991

Aspartate-like immunoreactivity in primary afferent neurons.

There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia. Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy; the great majority of cells immunopositive for aspartate were small (15-30 microns in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors. This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.

Afferent Pathways↗
PubMed1996

Complement 5a controls motility of murine microglial cells in vitro via activation of an inhibitory G-protein and the rearrangement of the actin cytoskeleton.

Microglial cells respond to most pathological events by rapid transformation from a quiescent to an activated phenotype characterized by increased cytotoxicity and motile activity. To investigate the regulation of microglial motility by different inflammatory mediators, we studied cultured murine microglia by time-lapse video microscopy and a computer-based motility assay. Microglial cells exhibited a high resting motility. The acute application of complement 5a (C5a) immediately induced intense ruffling of microglial membranes followed by lamellipodia extension within few seconds, while formyl-Met-Leu-Phe-OH, bacterial endotoxin (lipopolysaccharide) or inflammatory cytokines did not increase motility. This process was accompanied by a rapid rearrangement of the actin cytoskeleton as demonstrated by labelling with fluorescein isothiocyanate-phalloidin and could be inhibited by cytochalasin B. A GTP-binding protein was involved in the signal cascade, since pertussis toxin inhibited motility and actin assembly in response to C5a. Chemotactic migration in a gradient of C5a was also completely blocked by pertussis toxin and cytochalasin B. The C5a-induced motility reaction was accompanied by an increase in intracellular calcium ([Ca2+]i) as measured by a Fluo-3 based imaging system. Ca2+ transients were, however, not a prerequisite for triggering the increase in motility; motility could be repeatedly evoked by C5a in nominally Ca(2+)-free solution, while Ca2+ signals occurred only upon the first stimulation. Moreover, conditions mimicking intracellular Ca2+ transients, like incubation with thapsigargin or Ca2+ ionophore A23187, were not able to induce any motility reaction, suggesting that Ca2+ transients are not necessary for, but are associated with, microglial motility. Motile activity was shown to be restricted to a defined concentration range of [Ca2+]i as revealed by lowering [Ca2+]i with BAPTA-AM or increasing [Ca2+]i with A23187. Since complement factors are released at pathological sites, this signal cascade could serve to increase motility and to direct microglial cells to the lesioned or damaged area by means of a G-protein-dependent pathway and via the rearrangement of the actin cytoskeleton.

Actins↗
PubMed1996

Tonic protein kinase C-mediated vasoconstriction is unmasked when nitric oxide synthase is inhibited in cerebral microvessels.

Recent evidence indicates that nitric oxide participates in the modulation of vascular tone in a variety of vascular beds, including the parenchymal microvasculature of the brain. The present study examined the role of protein kinase activity in the induction and maintenance of the contractile response when endogenous nitric oxide production is inhibited in parenchymal microvessels of the rat hippocampus. Microvessels in in vitro slices of the hippocampus were monitored using computer-assisted video microscopy. The effects of inhibitors of two kinases, protein kinase C and calcium/calmodulin-dependent protein kinase, on the vasoconstrictor response to NG-nitro-L-arginine (L-NNA) were investigated. The resting luminal diameter of the microvessels examined in this study ranged from 9 to 29 microns. Addition of 100 microM L-NNA to the medium superfusing the slice constricted microvessels by 38.8 +/- 0.6%. The addition of protein kinase inhibitors reversed this constriction in a dose-dependent manner. H-7 (50 microM), a relatively non-selective protein kinase C inhibitor, elicited an 81.4 +/- 10.0% reversal of the L-NNA-induced constriction. Bisindolylmaleimide (5 microM), a selective protein kinase C inhibitor, reversed the constriction by 69.1 +/- 13.7%. KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II, elicited a smaller yet statistically significant reversal of 17.1 +/- 5.1%. Pretreatment with H-7 or bisindolyl-maleimide blocked the LNNA-induced constriction entirely, while KN-62 did not significantly inhibit the response. These findings indicate that the contractile response observed upon removal of endogenous nitric oxidergic vasodilation is mediated by protein kinase activity, and the contribution of protein kinase C to this effect is greater than that of calcium/calmodulin-dependent protein kinase II. The results suggest that a tonic nitric oxidergic influence serves to mask the potential for protein kinase C-mediated vasoconstriction in cerebral microvessels.

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