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PubMed2004

Adenosine's role in hypercapnia-evoked cerebral vasodilation in the rat.

Carbon dioxide induces a rapid dilation of cerebral arterioles, enabling local blood flow to match increasing metabolic requirements of tissue. Amongst the vasodilatory substances released by cortical tissue in response to CO2 are adenosine and nitric oxide. Here we report that selective adenosine A(2A) receptor antagonists, applied topically using a rat cortical window technique, significantly depressed the CO2-evoked increase in arteriolar diameter, measured using video microscopy, as well as attenuating the CO2 and pH reactivity of the cortical arterioles. Two non-selective inhibitors of nitric oxide synthase also significantly depressed the hypercapnia-evoked increase in arteriolar diameter.

Adenosine↗
PubMed2006

Erythrocyte exit: Out, damned merozoite! Out I say!

A new study has combined video microscopy with fluorescent labeling of host and parasite membranes to follow Plasmodium falciparum merozoites as they exit their host erythrocyte. The result has yielded some arresting images, which make compelling viewing irrespective of whether or not you have an interest in cell motility in general or P. falciparum erythrocyte exit in particular. Moreover, this work injects important new insights into the long-running debate about the biological mechanisms that underpin merozoite release.

Animals↗
PubMed2000

Intestinal restitution: progression of actin cytoskeleton rearrangements and integrin function in a model of epithelial wound healing.

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (alpha6beta4, alpha6beta1, and alpha3beta1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. alpha6beta4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the alpha6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha6 subunit inhibit lamellae formation, indicating that redistribution of the alpha6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha3beta1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The alpha6 integrins, particularly alpha6beta4, help contain retraction and both the alpha6 integrins and alpha3beta1 integrin contribute to lamellae formation.

Actins↗
PubMed1999

Comparative analysis of different methodological approaches to the in vitro study of drug-induced apoptosis.

Apoptosis is a dynamic process in which a characteristic morphological or biochemical event used in an assay as a specific marker of apoptosis may be observed over a limited period of time. Asynchronous involvement of cells in apoptosis results in different proportions of apoptotic cells with blebbed membrane, broken nuclei, modified mitochondrial units or fragmented DNA coexisting in the culture at any single moment. Thus, depending on the method used, the extent of apoptosis determined in the same cell population may vary. In the present study, a microculture kinetic (MiCK) assay was used to monitor apoptosis in HL-60 cells exposed to 1, 2.5, 5, 10, and 20 micromol/L etoposide and cisplatin. Both the extent and timing of apoptotic responses were dependent on the drug and drug concentration. Time-lapse video microscopy (TLVM), flow cytometry analysis of the light scattering properties of cells, morphological studies of Giemsa-stained cells, annexin V binding, and DNA fragmentation assays were performed at multiple times of cell exposure to 10 micromol/L etoposide and 5 micromol/L cisplatin. Steep linear increases in optical density, indicating apoptosis in the MiCK assay, correlated with both linear increases in the proportion of cells with plasma membrane blebbing in TLVM and with increased side scattering properties of apoptotic cells in flow cytometry. During a 24-hour culture period, the MiCK assay and TLVM provided multiple consecutive appraisals of nondisturbed cell microcultures at intervals of 5 and 2.5 minutes, respectively, and thus could be considered as real time kinetic assays. With the three endpoint assays, each of which was applied 12 times at 2-hour intervals, maximum apoptotic responses varied from 22.5 to 72% in etoposide-treated cells and from 30 to 57% in cisplatin-treated cells. With the annexin V binding assay, maximum apoptosis could always be detected 4 to 5 hours earlier than it was seen in Giemsa-stained preparations and 8 hours earlier than it was detected by measuring of DNA fragmentation. Values of the maximum extent of apoptosis varied, being the lowest with annexin V and the greatest with DNA fragmentation assays. The best correlations of both extent and timing of apoptosis were observed between the MiCK, TLVM, and morphological assays. In conclusion, both a maximum apoptotic response and the time at which it was achieved are the obligatory requirements for determining the apoptosis-inducing potency of an agent and for comparing results of studies performed in different laboratories.

Annexin A5↗
PubMed2000

The effects of various organ preservation solutions on hepatocyte membrane potentials, intracellular calcium concentrations, and outcome following liver transplantation.

BACKGROUND: Hepatocyte membrane potential differences (PDs) may be altered by the preservation solutions used in liver transplantation. Such alterations could impact on the survival of the donor liver, extent of biochemical injury, and flux of important ionic compounds. The purpose of the present study was to document these outcomes in the presence of four different preservation solutions. METHODS: Livers of adult male Sprague-Dawley rats (N = 3 to 4 per group) were impaled with intracellular microelectrodes prior to and at various time periods for 6 hours following complete hepatic resection. Just prior to resection, each liver was perfused with preservation solutions associated with high (normal saline [NS]), moderate (Euro-Collins [EC]), and low (University of Wisconsin solution [UW]) risks of reperfusion injury. RESULTS: Baseline (in situ) PDs were similar in all groups (-37 +/- 4 mV, mean +/- SD). Ten minutes postresection, hepatic PDs were as follows: NS, -23.8 +/- 3.5 mV; EC, -11.4 +/- 0.4 mV; and UW, -8.7 +/- 0.3 mV (P <0.01 for all groups). Maximum depolarization occurred at 6 hours postresection (NS, -8.1 +/- 1.1 mV; EC, -7.7 +/- 1.3 mV; and UW, -8.6 +/- 1.0 mV). To determine whether these changes are of pathophysiologic importance, the NS solution was modified (addition of 0.1% ethanol) to achieve similar PD changes as those observed with UW. Liver transplants were then performed where the donor livers had been perfused and preserved for 6 hours with either NS or the modified NS (MNS) solution. Posttransplant (10 day) survival was 1 of 6 (17%) in the NS group and 4 of 6 (67%) in the MNS group (P <0.05). Regarding the effects of PD changes on ionic flux, intracellular calcium levels were documented for up to 4 hours by fluorescence video microscopy using Fura-2 in isolated hepatocytes exposed to NS, UW, and MNS solutions. Intracellular calcium levels were similar in all solutions at each time point studied. CONCLUSIONS: The results of this study indicate that hepatocytes undergo prompt and marked depolarization following hepatic resection, and the extent of the depolarization correlates with survival following transplantation.

Adenosine↗
PubMed1998

Normothermic versus hypothermic hyperkalemic cardioplegia: effects on myocyte contractility.

BACKGROUND: This study was designed to determine the effects of prolonged hyperkalemic cardioplegic arrest under normothermic or hypothermic conditions with respect to left ventricular myocyte contractile performance and beta-adrenergic responsiveness. METHODS: Isolated left ventricular porcine myocytes were randomly assigned to one of three groups: (group 1) normothermic control, (group 2) hypothermic cardioplegic arrest, or (group 3) normothermic cardioplegic arrest. Myocyte contractility was evaluated by high-speed video microscopy at baseline and after beta-adrenergic stimulation with isoproterenol (25 nmol/L). RESULTS: Myocyte velocity of shortening was decreased after both hypothermic and normothermic cardioplegic arrest (68 +/- 2 and 69 +/- 2 microns/s, respectively) compared with normothermic control values (96 +/- 2 microns/s; p < 0.05). This relative reduction in baseline contractile function was equivalent in both cardioplegia groups (p = 0.5356). With beta-adrenergic stimulation, myocyte velocity of shortening was 186 +/- 4 microns/s in the hypothermic and 176 +/- 3 microns/s in the normothermic cardioplegia groups (p = 0.0563). However, myocyte contractility with beta-adrenergic stimulation was reduced in both cardioplegia groups compared with normothermic controls (205 +/- 4 microns/s; p < 0.05, respectively). CONCLUSIONS: Hyperkalemic cardioplegic arrest under either normothermic or hypothermic conditions resulted in an equivalent reduction in baseline myocyte contractile function with reperfusion/rewarming. Hypothermic cardioplegic arrest may have provided mild protective effects on beta-adrenergic responsiveness. Nevertheless, these results suggest that an important contributory factor for diminished myocyte contractility after simulated cardioplegic arrest was prolonged exposure to a hyperkalemic environment.

Adrenergic beta-Agonists↗
PubMed1999

Cellular and molecular therapeutic targets for treatment of contractile dysfunction after cardioplegic arrest.

Transient left ventricular (LV) dysfunction can occur after hypothermic hyperkalemic cardioplegic arrest. This laboratory has developed an isolated LV myocyte system of simulated cardioplegic arrest and rewarming in order to examine cellular and molecular events that may contribute to the LV dysfunction after cardioplegic arrest. Contractile function was examined using high-speed video microscopy after reperfusion and rewarming. After cardioplegic arrest and reperfusion, indices of myocyte contractility were reduced by over 40% from normothermic control values. The capacity of the myocyte to respond to an inotropic stimulus was examined through beta-adrenergic receptor stimulation with isoproterenol. After cardioplegic arrest, the contractile response to isoproterenol was reduced by over 50% from normothermic values. The next series of studies focused upon preventing these changes in myocyte contractile processes after cardioplegic arrest. First, the cardioplegic solutions were augmented with adenosine or an ATP-sensitive potassium channel opener, aprikalim. Both adenosine and aprikalim augmentation significantly improved myocyte function compared with cardioplegia alone values. A potential intracellular mechanism for the protective effects of either adenosine or the ATP-sensitive potassium channel is the activation of protein kinase C (PKC). A brief period of PKC activation before cardioplegic arrest provided protective effects on myocyte contractility with subsequent reperfusion and rewarming. In another set of studies, the potential protective effects of the active form of thyroid hormone (T3) were examined. In myocytes pretreated with T3, myocyte contractile function and beta-adrenergic responsiveness were significantly improved after hypothermic cardioplegic arrest and rewarming. Thus, endogenous means of providing improved myocardial protection during prolonged cardioplegic arrest can be achieved through a brief period of PKC activation or pretreatment with T3. Future studies, which more carefully deduce the basis for these pretreatment effects, will likely yield novel methods by which to protect myocyte contractile processes during cardioplegic arrest.

Adenosine↗
PubMed2001

Cholesterol stabilizes hemifused phospholipid bilayer vesicles.

Cholesterol was found to inhibit full fusion of oppositely charged phospholipid bilayer vesicles by stabilizing the contacting membranes at the stage of the hemifused intermediate. Vesicles of opposite charge containing different amounts of cholesterol were prepared using cationic (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and anionic (dioleoylphosphatidylglycerol) phospholipids. Pairwise interactions between such vesicles were observed by fluorescence video microscopy in real time after electrophoretically maneuvering the vesicles into contact. Hemifusion accounted for more than 80% of the observed events when the vesicles contained 33-50 mole% cholesterol. In contrast, vesicles containing only a small proportion of cholesterol (</=10 mole%), underwent full fusion in approx. 70% of the interactions monitored. The role of cholesterol is explained both as favoring the formation of the hemifused intermediate according to the adhesion-condensation mechanism of bilayer fusion and as disfavoring the transition from hemifusion to full fusion on the basis of reduced tension in the vesicle bilayers.

Cholesterol↗
PubMed2003

Kinetically differentiating influenza hemagglutinin fusion and hemifusion machines.

Membrane fusion mediated by influenza virus hemagglutinin (HA) yields different phenotypes depending on the surface density of activated HAs. A key question is whether different phenotypes arise from different fusion machines or whether different numbers of identical fusion machines yield different probabilistic outcomes. If fusion were simply a less probable event than hemifusion, requiring a larger number of identical fusion machines to occur first, then two predictions can be made. First, fusion should have a shorter average delay time than hemifusion, since there are more machines. Second, fusion should have a longer execution time of lipid mixing after it begins than hemifusion, since the full event cannot be faster than the partial event. Using a new automated video microscopy technique, we simultaneously monitored many HA-expressing cells fusing with erythrocytes and identified individual cell pairs with either full or only partial redistribution of fluorescent lipids. The full lipid mixing phenotype also showed contents mixing, i.e., fusion. Kinetic screening of the digitized fluorescence data showed that the execution of lipid mixing after the onset is faster for fusion than hemifusion. We found no correlation between the delay times before the onset of lipid mixing and the final fusion phenotype. We also found that the execution time for fusion was faster than that for hemifusion. Thus, we provide the first experimental evidence for fusion and hemifusion arising from different machines.

Biophysical Phenomena↗
PubMed1995

Local positive feedback by calcium in the propagation of intracellular calcium waves.

In many types of eukaryotic cells, the activation of surface receptors leads to the production of inositol 1,4,5-trisphosphate and calcium release from intracellular stores. Calcium release can occur in complex spatial patterns, including waves of release that traverse the cytoplasm. Fluorescence video microscopy was used to view calcium waves in single mouse neuroblastoma cells. The propagation of calcium waves was slowed by buffers that bind calcium quickly, such as BAPTA, but not by a buffer with slower on-rate, EGTA. This shows that a key feedback event in wave propagation is rapid diffusion of calcium occurring locally on a scale of < 1 micron. The length-speed product of wavefronts was used to determine that calcium acting in feedback diffuses at nearly the rate expected for free diffusion in aqueous solution. In cytoplasm, which contains immobile Ca2+ buffers, this rate of diffusion occurs only in the first 0.2 ms after release, within 0.4 micron of a Ca2+ release channel mouth. Calcium diffusion from an open channel to neighboring release sites is, therefore, a rate-determining regenerative step in calcium wave propagation. The theoretical limitations of the wave front analysis are discussed.

Animals↗
PubMed1995

Dynamics of photoinduced cell plasma membrane injury.

We have developed a video microscopy system designed for real-time measurement of single cell damage during photolysis under well defined physicochemical and photophysical conditions. Melanoma cells cultured in vitro were treated with the photosensitizer (PS), tin chlorin e6 (SnCe6) or immunoconjugate (SnCe6 conjugated to a anti-ICAM monoclonal antibody), and illuminated with a 10 mW He/Ne laser at a 630 nm wavelength. Cell membrane integrity was assessed using the vital dye calcein-AM. In experiments in which the laser power density and PS concentration were varied, it was determined that the time lag before cell rupture was inversely proportional to the estimated singlet oxygen flux to the cell surface. Microscopic examination of the lytic event indicated that photo-induced lysis was caused by a point rupture of the plasma membrane. The on-line nature of this microscopy system offers an opportunity to monitor the dynamics of the cell damage process and to gain insights into the mechanism governing photolytic cell injury processes.

Cell Line↗
PubMed2003

Cortical electrical stimulation alters erythrocyte perfusion pattern in the cerebral capillary network of the rat.

The effect of direct cortical electrical stimulation on the pattern of erythrocyte perfusion in the capillary network of the rat cerebral cortex was studied by fluorescence intravital video-microscopy. The movement of fluorescently labeled red blood cells (FRBCs) in individual capillaries 50-70 microm subsurface in the dorsal somatosensory cortex was visualized using a closed cranial window. Cortical stimulation electrodes were placed on opposite sides of the window. FRBC velocity (mm/s) and supply rate (cells/s) were measured in 51 capillaries from six rats before and during electrical stimulation of increasing intensities (15-s trains of 3-Hz, 3-ms, 0.5-5.0-mA, square pulses). FRBC velocity, supply rate, and the instantaneous capillary erythrocyte content (lineal cell density, LCD, cells/mm) increased with the stimulation current and reached maxima of 110, 160 and 33% above control, respectively. Capillaries with low resting velocity showed a greater response than those with high resting velocity. The fraction of capillaries in which FRBC velocity increased was not constant, but increased with the stimulation current, as did the magnitude of the velocity change in these capillaries. A few capillaries showed a negative FRBC velocity response at stimulations <4 mA. These results suggest that a robust rise in the fraction of responding (engaged) capillaries and a smaller rise in the capillary LCD contribute to neuronal activation-induced cortical hyperemia. Thus, capillary engagement and erythrocyte recruitment appear to represent important components of the cortical functional hyperemic response. These results provide insight into some of the specific hemodynamic changes associated with functional hyperemia occurring at the capillary level.

Animals↗
PubMed1999

Increased myogenic tone in skeletal muscle arterioles of diabetic rats. Possible role of increased activity of smooth muscle Ca2+ channels and protein kinase C.

OBJECTIVE: The diabetes mellitus-induced microangiopathy is still not clearly characterized. In this study we aimed to elucidate the effect of streptozotocin (STZ)-induced diabetes on myogenic response of isolated rat skeletal muscle arterioles and the mechanisms responsible for its alterations. METHODS: Male rats were divided into two groups: (1) control rats (C, plasma glucose: 6.4 +/- 0.5 mmol/l, n = 40) (2) diabetic rats (DM, 65 mg/kg STZ i.v., plasma glucose: 25.7 +/- 0.7 mmol/l, n = 40). Changes in diameter of isolated, cannulated gracilis skeletal muscle arterioles (approximately 130 microns in diameter) were measured by video-microscopy. RESULTS: Step increases in perfusion pressure (PP; from 10 to 140 mmHg) elicited significantly greater constrictions in DM than in C gracilis arterioles, in the presence of the endothelium (E). Also, a step increase in PP (from 40 to 100 mmHg) elicited greater and faster constrictions in DM vs. C arterioles. There were no significant differences in the pressure-passive diameter (in Ca2+ free solution) curves of arterioles. Dilations to acetylcholine were impaired in arterioles of DM as compared to those of C rats (EC50, C: 4.0 +/- 0.9 x 10(-9) mol/l, DM: 4.8 +/- 2.0 x 10(-8) mol/l (p < 0.01), and unaffected by inhibition of nitric oxide synthesis with L-NNA (10(-4) mol/l). Arteriolar constrictions to norepinephrine (NE) were significantly greater in DM compared to those of C rats (EC50, C: 6.2 +/- 0.6 x 10(-7) mol/l, DM: 8.0 +/- 2.0 x 10(-8) mol/l, p < 0.01) both in the presence and absence of E. In the absence of the E, constrictions to increases in pressure, or Ca2+ (0.25-7.5 mmol/l), or the voltage-dependent Ca(2+)-channel agonist Bay K 8644 (EC50; DM: 4.2 +/- 1.5 x 10(-10) mol/l, C: 1.7 +/- 0.8 x 10(-9) mol/l, p < 0.05) or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA, EC50; DM: 6 +/- 2 x 10(-9) mol/l, C: 2 +/- 1 x 10(-8) mol/l, p < 0.05) were significantly greater in arterioles of DM compared to those of C rats. CONCLUSION: The novel findings of our study are that in diabetes mellitus the myogenic response of rat skeletal muscle arterioles is enhanced, which seems to be independent from the impaired endothelial function present simultaneously, and likely due to the increased activity of voltage-dependent Ca2+ channels and/or upregulation of protein kinase C in arteriolar smooth muscle.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2003

Myelin growth and initial dynamics.

During the dissolution of solid surfactants in water, various types of nonequilibrium microstructures have been observed. The most important one is the myelin growth which can be observed when some poorly water soluble surfactants such as phosphatidylcholine (PC), Aerosol-OT (AOT), etc. are contacted with water. In this study initial myelin growth for a period of 2-4 s is studied both qualitatively as well as quantitatively in all the directions with respect to water flow in a PC system using digital video microscopy. Further, overall myelin growth is studied by means of optical microscopy to understand the effect of distance between cover slip and glass slide on myelin growth. Experiments are also performed to study effect of additives (silica) to lamellar phase on diffusion coefficients. It has been observed that the presence of silica particles causes extensive coiling of myelin structures. The mechanism of water transport into the lamellar phase during myelin growth is investigated by using silica in a colloidal range as dopant material.

Dioctyl Sulfosuccinic Acid↗
PubMed1995

Stretch-activated channel blockers modulate cell volume in cardiac ventricular myocytes.

Stretch-activated channels (SAC) are postulated to regulate cell volume. While this hypothesis is appealing, direct evidence is lacking. Using digital video microscopy, we found that pharmacological blockade of SACs alters the cell volume of isolated rabbit ventricular myocytes during hypoosmotic stress. Under control conditions, relative cell volume increased from 1.0 to 1.311 +/- 0.019 after 10 min in 195 mosmol/l solution. The cation SAC blocker gadolinium (Gd3+; 10 microM) reduced the amount of swelling in hypoosmotic solution by 24% and induced a regulatory volume decrease otherwise not observed. In contrast, the anion SAC blocker 9-anthracene carboxylic acid (9-AC; 1 mM) increased swelling by 44% under the same conditions. Based on the direction of SAC currents, Gd3+ and 9-AC are expected to have opposite effects on cell volume. Furthermore, Gd3+ and 9-AC changed cell volume by only approximately 2% in isosmotic solutions when SACs are expected to be closed. This supports the idea that Gd3+ and 9-AC affect stretch-activated transport processes. In contrast, omitting bath Ca2+ did not alter cell volume under iso- or hypoosmotic conditions suggesting stretch-activated Ca2+ influx is not important in setting cell volume. Not all channels can affect cell volume. Opening ATP-sensitive K+ channels with aprikalim (100 microM) or blocking them with glibenclamide (1 microM) did not alter cell volume under isosmotic or hypoosmotic conditions. These data support the idea that SACs are involved in cardiac cell volume regulation.

Animals↗
PubMed2003

Ultrasound measurement of aortic diameters in rodent models of aneurysm disease.

BACKGROUND: This investigation was undertaken to evaluate transabdominal ultrasound (US) measurements of aortic diameters in rats and mice as a complementary method to video microscopy (VM), the current standard for assessing the diameter of rodent aortas. METHODS: Aortic diameters were measured in 64 rats (n = 132 sets) and 12 mice (n = 36 sets) following experimental induction of aortic aneurysms. Diameters were measured at the renal vein, midinfrarenal aorta, and aortic bifurcation. RESULTS: In the rat, anteroposterior (AP) US measurements were closely correlated with transverse VM measurements, with correlation coefficients ranging from 0.66 to 0.77 (P < 0.0001) for axial US images and 0.58 to 0.63 (P < 0.0001) for sagittal US images. In the mouse, significant correlation coefficients were 0.57 (P < 0.001) near the renal vein and 0.44 (P = 0.007) at the midinfrarenal aorta. Aortic diameters increased significantly with increasing animal age and weight (R = 0.40, P = 0.003 at the renal vein, R = 0.29, P = 0.04 in the midinfrarenal aorta, and R = 0.39, P = 0.004 at the aortic bifurcation), suggesting that weight matched rodents must be used to define aortic dimensions in treatment groups as opposed to repeated comparisons with baseline measurements in a growing rat. CONCLUSION: Noninvasive aortic US measurements throughout the course of a rodent study of aneurysmal disease provide a practical alternative to VM for the repeated determinations of aortic diameters.

Animals↗
PubMed2000

Rabbit ventricular myocyte volume changes as a direct result of crystalloid cardioplegia in congestive heart failure induced by aortic regurgitation.

OBJECTIVES: We hypothesized that the cell volume of ventricular myocytes isolated from hearts in volume-overload congestive failure would respond differently to hypothermic cardioplegia than would sham-operated cohorts. METHODS: Adult rabbits underwent either valvotomy and aortic regurgitation-induced heart failure or sham surgery. Congestive failure was confirmed clinically and by means of echocardiography. Cell volumes of isolated myocytes were measured by digital video microscopy. After equilibration in 37 degrees C physiologic solution, cells were suprafused with 9 degrees C standard or low-Cl(-) St Thomas' Hospital solution followed by reperfusion in 37 degrees C physiologic solution. RESULTS: Exposure to cold St Thomas' Hospital solution for 20 minutes caused sham myocytes to swell by 8% (n = 9); cell volumes fully recovered on normothermic reperfusion. In contrast, congestive failure myocytes (n = 9) maintained their cell volume in cold St Thomas' Hospital solution and during reperfusion. Lowering the [K(+)][Cl(-)] product of St Thomas' Hospital solution by partially replacing Cl(-) with an impermeant anion prevented cellular edema in the sham group (n = 8) but caused a 4% swelling in failure myocytes (n = 10) on reperfusion. Osmotically shrinking the failure cells (n = 9) converted their behavior to that of sham cells. CONCLUSIONS: In the absence of ischemia, congestive failure myocytes are less sensitive to cardioplegia-induced edema than sham cells. Low-Cl(-) cardioplegia, which prevents edema and protects the normal heart, induced swelling and may be detrimental in myopathic hearts. Differences in volume regulation in failure and sham myocytes may be due to activation of volume-sensitive channels that are turned off by osmotic shrinkage.

Animals↗
PubMed1995

Adenosine triphosphate-sensitive K+ channels mediate postcardioplegia coronary hyperemia.

The purpose of the present study was to examine the role of adenosine triphosphate-sensitive potassium channels in mediating the coronary hyperemic response after crystalloid cardioplegia. Thirteen pigs were placed on normothermic cardiopulmonary bypass support. Hearts were arrested with cold (4 degrees C) crystalloid ([K+] 25 mmol/L) cardioplegic solution for 60 minutes. In seven of these pigs, hearts were then reperfused for 60 minutes with warm blood, and the animal was separated from cardiopulmonary bypass. The in vivo responses to the intracoronary administration of the K+ adenosine triphosphate channel blocker glibenclamide (50 gm/kg per minute) or the K+ adenosine triphosphate channel opener pinacidil (2 gm/kg per minute) were evaluated before cardiopulmonary bypass (baseline) and after 2 minutes and 60 minutes of reperfusion in the cardioplegia-reperfusion group. Under baseline conditions, glibenclamide and pinacidil induced a respective decrease and increase in coronary blood flow and an increase and a decrease in coronary vascular resistance. Coronary responses to glibenclamide and pinacidil were markedly enhanced after 2 minutes or 60 minutes of postcardioplegia reperfusion. In vitro responses of coronary arterioles (90 to 180 microns) were examined in a pressurized, no-flow state with video microscopy. The contractile response of coronary arterioles to glibenclamide and the relaxation response to pinacidil were significantly enhanced 2 minutes or 60 minutes after reperfusion (all p < 0.05 versus control). The response to pinacidil was markedly inhibited by glibenclamide, which confirms these antagonistic effects on K+ adenosine triphosphate channels. Decreased tissue concentrations of adenosine triphosphate in the coronary arterial smooth muscle and myocardium were observed after cardioplegia and persisted for up to 60 minutes of reperfusion (both p < 0.05 versus control). These results suggest that coronary hyperemia associated with postischemic cardioplegia is mediated in part by activation of K+ adenosine triphosphate channels in the coronary microcirculation.

Adenosine Triphosphate↗