Technique for using video microscopy and indicator dilution for repeated measurements of cardiac output in small animals.

BACKGROUND: The authors developed an indicator dilution technique for small animals to repeatedly determine cardiac output and blood volume without cardiac instrumentation or blood sampling. METHODS: Observations were made in the hamster (N = 32, 70 mg/kg pentobarbital) cremaster using in vivo fluorescence videomicroscopy. Fluorescein isothiocyanate-conjugated bovine serum albumin (10 mg/ml) was injected as a bolus dose (right jugular) while video recording the light intensity in a 20-microm arteriole (intensified charge-coupled device [CCD] camera at fixed gain). The intensity signal was analyzed over time (background subtracted) and calibrated to the dye concentration. The ex vivo calibration was performed using a constant optical path length (20 microm) and a range of dye and hematocrit concentrations. In vivo tube hematocrit was determined using standard methods with fluorescently labeled erythrocytes. Thus, quenching of the fluorescence signal by hemoglobin was corrected for the calibration, and the plasma space in the arteriole was determined. The steady state dye concentration measured by the light intensity at 2 min was not different from the dye concentration found by direct spectrophotometric analysis of the plasma. RESULTS: Cardiac index was calculated as milliliters of blood per minute per kilogram body weight. The calculated cardiac index was 359 +/- 18 ml.min(-1).kg(-1), which is not different from the reported values for hamsters. Cardiac output was increased twofold when enough intravenous nitroprusside or nitroglycerine was injected to decrease mean arterial pressure from 90 to 70 mmHg. Cardiac output was elevated during dobutamine infusion (16 microg.kg(-1).min(-1)) and decreased during esmolol infusion (50, 75.kg(-1).min(-1)). Blood volume determined from the steady state dye concentrations was 6.2 +/- 0.5 ml/100 g body weight, within the normal range for hamsters. CONCLUSIONS: Fluorescent dye dilution and video microscopy can be used to repeatedly determine cardiac output or blood volume in small animals.

Localized adenovirus-mediated gene transfer into vascular smooth muscle in the hamster cheek pouch.

OBJECTIVE: Our purpose was to develop a method for adenovirus delivery to the hamster cheek pouch to experimentally target gene transfer in tissue used for microvascular studies. METHODS: Separate constructs were tested with transgenes for lacZ or green fluorescent protein (GFP) driven by three promoters: RSV, CMV, and SM22. With university approval, adenovirus was delivered in anesthetized (pentobarbital, 70 mg/kg) hamsters (n = 28) by using either a vascular systemic injection or tissue infiltration (interstitial space behind the pouch). During 3 days, animals receiving infiltration gained the expected weight, whereas those receiving vascular injection lost weight; no other behavior changes were noted. RESULTS: On day 3 postadenoviral delivery (infiltration), expression of lacZ (histology, beta-galactosidase) or GFP (fluorescence microscopy) was confirmed across the tissue (CMV and RSV promoters) and exclusively in vascular smooth muscle cells (specific SM22 promoter), without evidence of tissue inflammation. In vitro microvascular experiments verified normal responses in the cheek pouch of day 3 postadenoviral delivery animals. We tested local dilation to methacholine, adenosine, remote dilation to methacholine, adenosine, nitroprusside, and LM609 (alpha(v)beta3 integrin agonist), flow-dependent dilation, and flow recruitment. CONCLUSIONS: Thus, this method enables targeted, cell-specific gene transfer to one tissue important for microvascular studies, without significant systemic exposure and without adverse inflammation.

Increased flow precedes remote arteriolar dilations for some microapplied agonists.

This study asks which occurs first in time for remote responses: a dilation or a remote change in flow. Arteriolar diameter (approximately 20 microm) and fluorescently labeled red blood cell (RBC) velocity were measured in the cremaster muscle of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n = 51). Arterioles were locally stimulated for 60 s with micropipette-applied 10 microg/ml LM-609 (alpha(v)beta(3)-integrin agonist), 10(-3) M adenosine, or 10(-3) M 3-morpholinosydnonimine (SIN-1, nitric oxide donor) as remote response agonists or with 10(-3) M papaverine, which dilates only locally. Observations were made at a remote site 1,200 microm upstream. With LM-609 or adenosine, the RBC velocity increased first (within 5 s), and the remote dilation followed 5-7 s later. N-nitro-L-arginine (100 microM) blocked the LM-609 (100%) and adenosine (60%) remote dilations. SIN-1 induced a concurrent remote dilation and decrease in RBC velocity (approximately 10 s), suggesting the primary signal was to dilate. Papaverine had no remote effects. This study suggests that, although remote responses to some agonists are induced by primary signals to dilate, additionally, network changes in flow can stimulate extensive remote changes in diameter.

Flow-induced cytoskeletal changes in endothelial cells growing on curved surfaces.

OBJECTIVE: Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow-induced F-actin organization in endothelial cells. METHODS: Human umbilical vein endothelial cells were grown to confluence on curved or flat surfaces. Microchannels (curved surface, 10- to 30-microm radius) or parallel plate flow chambers were perfused (30 minutes to 6 hours) at physiological flow rates (wall shear stress 1 to 10 (dyn/cm2). RESULTS: On curved surfaces, the number of central F-actin stress fibers (for cells of equal area) decreased from 4.8+/-0.3 (mean +/- SE, n = 36) (static) to 0.9+/-0.5 per cell in perfused microchannels. Perfusion with 100 microM histamine prevented this response to flow (5.5+/-0.8 per cell, n = 12). Stress fibers were initially aligned with tile long axis of the microchannel at an angle of 9+/-0.7 degrees (static). With flow, alignment of the few remaining central F-actin stress fibers with respect to the long axis of the microchannel decreased to 19+/-4 degrees this was prevented by perfusion with histamine (5.6+/-1 degrees). The number of stress fibers per cell, for cells grown on flat surfaces (8.1+/-0.3, static, n = 36) was significantly greater than cells on curved surfaces, and did not change with flow (8.1+/-0.5 per cell, n = 6). On flat surfaces, the stress fiber orientation with respect to the longitudinal axis of the channel) was 42+/-1.4 degrees (static) and did not change with flow (38+/-4.2 degrees). CONCLUSIONS: Endothelial cells on curved growth surfaces respond to flow rapidly, with marked changes in F-actin central stress fiber formation. This implicates a tight relationship between cell shape and the environmental substrate, and suggests that the shape of the endothelial cell significantly impacts its ability to respond to its environment.

Predicted wall shear rate gradients in T-type arteriolar bifurcations.

The purpose of this study was to examine the theoretical impact of the local bifurcation geometry on the shear rate gradient in a divergent arteriolar-type bifurcation. Newtonian flow through an arteriolar bifurcation was modeled using 3-dimensional computational fluid dynamics (CFD). Branching angles of 30 degrees, 50 degrees, 70 degrees, 90 degrees, 110 degrees, 130 degrees, and 150 degrees were studied at a Reynolds number (Re) of 0.01 in seven separate models. Both the flow split (30%) and the branch to main vessel diameter ratio (4/5) were held constant. Velocity profiles were predicted to deviate significantly from a parabolic form, both immediately before and after the branch. This deviation was shown to be a function of the local bifurcation geometry of each model, which consisted of a branching angle and associated feed-branch intersection shape. Immediately before and after the branch, the shear rate along the lateral branching wall was predicted to exceed (5-fold) that calculated for fully developed flow in the feed. In vivo data were from the anesthetized (pentobarbital, 70 mg/kg) hamster cremaster muscle preparation. Red blood cells were used as flow markers in arteriolar branch points (n = 74) show that a significant gradient in shear rate occurs at the locations and branch shapes predicted by the computational model. Thus, for low Re divergent flow, the gradient in shear rate measured for non-Newtonian conditions, is approximated by a finite element fluid dynamics model of Newtonian flow.

Microcirculatory basis for nonuniform flow delivery with intravenous nitroprusside.

BACKGROUND: The purpose of this study was to determine the effects of systemic infusions of nitroglycerin and sodium nitroprusside on flow distribution and wall shear stress in the microcirculation. METHODS: With university approval, the cremaster muscle of 28 anesthetized (70 mg/kg pentobarbital given intraperitoneally) hamsters (Harlan Sprague Dawley: Syrian; weight, 121+/-11 g [mean +/- SDD) was observed using in vivo fluorescence microscopy. Arteriolar diameter, erythrocyte flux, and velocity were measured for a feed arteriole and its sequential branches. Observations were made during control (mean arterial pressure, 88+/-4 mm Hg) and after 30 min of intravenous delivery of sodium nitroprusside or nitroglycerin, titrated to decrease mean arterial pressure by 20 mm Hg. RESULTS: Sodium nitroprusside significantly dilated select upstream portions of the network (23+/-2.6 to 29+/-2.6 microm); no arterioles were dilated with nitroglycerin. Erythrocyte flux into the feed (i.e., inflow into the arteriolar network) and into the sequential branches (i.e., distribution within the network) were evaluated. With nitroglycerin, inflow decreased significantly from 1,560+/-335 to 855+/-171 cells/s, and flux into the branches decreased evenly. With sodium nitroprusside, inflow increased significantly to 2,600+/-918 cells/s, yet cells were "stolen" from upstream branches (a decrease from 425+/-67 to 309+/-87 cells/s in the first branch). Excess flow passed into a downstream "thorough-fare channel," significantly increasing flux from 347+/-111 to 761+/-246 cells/s. Wall shear stress decreased uniformly with nitroglycerin infusion, with a decrease in the feed from 8.8+/-2.5 to 6+/-1.7 dyn/cm2. With sodium nitroprusside, variable changes occurred that were location specific within the network. For instance, at the inflow point to the network, wall shear stress changed from 8.3+/-2.5 to 4.2+/-3.3 dyn/cm2. CONCLUSIONS: Nitroglycerin infusion promoted homogeneity of flow. Sodium nitroprusside significantly increased the heterogeneity of flow within this arteriolar network; an anatomic location for steal induced by sodium nitroprusside is identified.

Network vascular communication initiated by increases in tissue adenosine.

Vascular communication of vasomotor signals appears to coordinate the distribution of tissue blood flow. This study was performed to determine whether elevated tissue concentrations of adenosine or nitric oxide could induce vascular communicating signals. To test this, remote arteriolar responses were tested when drugs were applied either directly to an arteriole ( approximately 20 microm diameter), or into the tissue in a region (with no vessels over 10 microm in diameter) that was 500 microm away from the arteriole and that bore no defined relationship to the flow path of the remote arteriole. In anesthetized hamster cheek pouch (n = 25), or cremaster muscle (n = 10), remote arteriolar responses were measured in response to nitric oxide (NO) donors (10(-5) to 10(-3) M), adenosine (10(-5) to 10(-3) M), or papaverine (10(-5) to 10(-2) M) applied for 40-120 s. Papaverine caused no remote response when applied directly while adenosine and NO donors caused similar, late-onset (10-20 s), dose-dependent, remote responses in both preparations. Remarkably however, only adenosine initiated a consistent remote arteriolar dilation when applied to the tissue site. Thus, increases in tissue adenosine may be critical for vascular communication of metabolic demands without regard to the specific blood flow path.

Conducted signals within arteriolar networks initiated by bioactive amino acids.

Our purpose was to determine the specificity of L-arginine (L-Arg)-induced conducted signals for intra- vs. extracellular actions of L-Arg. Diameter and red blood cell velocities were measured for arterioles [18 +/- 1.6 (SE) micrometer] in the cremaster muscle of pentobarbital sodium-anesthetized (Nembutal, 70 mg/kg) hamsters (n = 53). Remote (conducted) responses were viewed approximately 1,000 micrometer upstream from the local (micropipette) application. Six amino acids were tested: L-arginine, L-cystine, L-leucine, L-lysine, L-histidine, and L-aspartate (100 microM each). Only L-Arg induced a remote dilation; L-lysine and L-aspartate had no effect, and the others each induced a significant remote constriction. There is a second conducted signal initiated by L-arginine that preconditions the arteriolar network and upregulates a direct response of L-arginine to dilate the remote site. This was blocked by inhibition of L-arginine uptake at the local (preconditioning) site (100 microM L-histidine or 1 mM phenformin). Arginine-glycine-aspartate (100 microM)-induced remote dilations (+3. 2 +/- 0.3 micrometer) were not mimicked by a peptide control and were prevented by anti- integrin alphav monoclonal antibody. Remote dilations were greater in animals with a higher wall shear stress for arginine-glycine-aspartate (r2 = 0.92) but not for L-arginine (r2 = 0.12). Thus L-arginine initiates separate conducted signals related to system y+ transport, integrins, and baseline flow.

Shear stress gradient over endothelial cells in a curved microchannel system.

Our purpose was to test a scale model of the microcirculation by measuring the shear forces to which endothelial cells were exposed, and comparing this to computer simulations. In vitro experiments were performed to measure the 2-dimensional projected velocity profile along endothelial cell lined microchannels (D-shaped, 10-30 microns radius, n = 15), or in microchannels without endothelial cells (n = 18). Microchannels were perfused with fluorescently labeled microspheres (0.5 micron dia., < 1%) suspended in cell culture media. The velocity of individual microspheres was obtained off-line (videorecording), using an interactive software program; velocity was determined as the distance traveled in one video field (1/60 s). Mass balance was verified in the microchannels by comparing the microsphere velocities to the perfusion pump rate. In confluent endothelial cell lined microchannels, a velocity profile was obtained as microspheres passed an endothelial cell nucleus (identified by fluorescent dye), and again, for a paired region 100 microns away without nuclei (cytoplasm region). The velocity profile was significantly shifted and sharpened by the endothelial cell nucleus, as anticipated. Over the nucleus, data are consistent with a normal sized nucleus extending into the lumen, further confirming that this scale model can be used to determine the wall shear stress to which endothelial cells are exposed. Using the experimental bulk phase fluid parameters as boundary conditions, we used computational fluid dynamics (CFD) to predict the expected wall shear stress gradient along an endothelial cell lined D-shaped tube. The wall shear stress gradient over the nucleus was 2-fold greater in the radial versus axial directions, and was sensitive to lateral versus midline positioned nuclei.

Endothelial cell dilatory pathways link flow and wall shear stress in an intact arteriolar network.

Our purpose was to determine whether the endothelial cell-dependent dilatory pathways contribute to the regulation of flow distribution in an intact arteriolar network. Cell flow, wall shear stress (T omega), diameter, and bifurcation angle were determined for four sequential branches of a transverse arteriole in the superfused cremaster muscle of pentobaribtal sodium (Nembutal, 70 mg/kg)-anesthetized hamsters (n = 51). Control cell flow was significantly greater into upstream than into downstream branches [1,561 +/- 315 vs. 971 +/- 200 (SE) cells/s, n = 12]. Tissue exposure to 50 microM N omega-nitro-L-arginine + 50 microM indomethacin (L-NNA + Indo) produced arteriolar constriction of 14 +/- 4% and decreased flow into the transverse arteriole. More of the available cell flow was diverted to downstream branches, yet flow distribution remained unequal. Control T omega was higher upstream than downstream (31.3 +/- 6.8 vs. 9.8 +/- 1.5 dyn/cm2). L-NNA + Indo decreased T omega upstream and increased T omega downstream to become equal in all branches, in contrast to flow. To determine whether constriction in general induced the same changes, 5% O2 (8 +/- 4% constriction) or 10(-9) M norepinephrine (NE; 4 +/- 3% constriction) was added to the tissue (n = 7). With O2, flow was redistributed to become equal into each branch. With NE, flow decreased progressively more into the first three branches. The changes in flow distribution were thus predictable and dependent on the agonist. With O2 or NE, the spatial changes in flow were mirrored by spatial changes in T omega. Changes in diameter and in cell flux were not related for L-NNA + Indo (r = 0.45), O2 (r = 0.07), or NE (r = 0.36). For all agonists, when the bifurcation angle increased, cell flow to the branch decreased significantly, whereas if the angle decreased, flow was relatively preserved; thus active changes in bifurcation angle may influence red cell distribution at arteriolar bifurcations. Thus, when the endothelial cell dilatory pathways were blocked, the changes in flow and in T omega were uncoupled; yet when they were intact, flow and T omega changed together.

A system for culture of endothelial cells in 20-50-microns branching tubes.

OBJECTIVE: To construct an in vitro endothelial cell culture system which would mimic the geometry and hemodynamic conditions of the arteriolar microcirculation. METHODS: Using a photolithography technique, semicircular channels (20-50 microns in diameter) were etched in mirror-image patterns on pairs of borosilicate microscope slide glass. One-half of each plate pair was predrilled with perfusion port holes at funnel-shaped fluid entrance regions. The perfusion system was constructed of micropipette glass and Teflon tubing, and imbedded in Sylgard. Two types of endothelial cells were grown to confluence within the half-channels: rabbit lung microvascular endothelial cells (a gift of Dr. M.E. Gerritsen, Miles Inc.) and human umbilical vein endothelial cells. After the cells were confluent, the two mirror images were aligned and clamped together to form a complete branching system of tubes lined with endothelial cells. RESULTS: This cell culture system can be perfused at physiological flow rates corresponding to wall shear stress values in the range 0.03-48 dyn/cm2. The fluid velocity profiles can be measured in this system by tracking the velocity and flow paths of 0.5-micron fluorescently labeled microspheres. Endothelial cells which grow within the channel exhibit F-actin alignment along the long axis of the channel by 3 days after seeding. Scanning electron micrographs indicate that 4 hr after seeding, endothelial cells commonly form cellular projections extending across the half-channel; by 5 days after seeding, the projections appear to have flattened out along the bottom of the channel. CONCLUSIONS: An in vitro endothelial cell culture system was constructed which mimics the geometry and hemodynamics conditions of resistance arterioles. This system can be used to examine endothelial cell responses to flow and flow gradients under defined and controllable conditions which mimic the arteriolar microcirculation.

Energy optimization and bifurcation angles in the microcirculation.

Our purpose was to examine the relationship between bifurcation angle and energy optimization in the arteriolar microcirculation. We measured bifurcation angles and diameters for sequential branches along a third-order feed arteriole (25 microns) in the superfused cremaster muscle of anesthetized (pentobarbital, 70 mg/kg) Golden hamsters (N = 51). Predicted bifurcation angles were calculated using the diameter data in a model designed to minimize total energy or using four different models each designed to minimize a specific energy cost (vessel wall surface area, vascular volume, wall shear stress, power losses), these models each assuming constant viscosity and that branching occurs with perfect space filling (i.e. junction exponent, x, = 3). The range of the predicted bifurcation angles for any model was small (+/- 10 degrees), and they were not different for the sequential junctions along the feed arteriole, where the observed angles significantly decreased in angle along the feed (first junction, 115 +/- 4.4 degrees; second, 88 +/- 5.2 degrees; third, 76 +/- 4.8 degrees; and last, 57 +/- 3.4 degrees). We next corrected for a nonconstant viscosity by using our in vivo tube hematocrit data and a published relationship among diameter, tube hematocrit, and apparent viscosity. Again assuming that x = 3, the total energy minimization model now predicted that the bifurcation angle was always obtuse and not different for the sequential branches along the feed arteriole (first, 125 +/- 3.3 degrees; second, 124 +/- 3.4 degrees; third, 120 +/- 6.6 degrees; and last, 132 +/- 2.7 degrees); the predicted angles were not correlated with the observed angles (r = 0.25). Using the geometric resistance (diameters) and the angles measured in vivo, and assuming constant viscosity, we next calculated the value of chi for each of the bifurcation junctions for each of the four models described above. The average value of x was not equal to 3 for any of the four models. The value of x decreased along the feed arteriole (first to last branch) from 2.7 +/- 0.26 to 1.6 +/- 0.22 (surface) and from 4.2 +/- 0.36 to 2.9 +/- 0.23 (volume), and x increased along the feed from 3.0 +/- 0.35 to 15.5 +/- 2.6 (shear stress) and from 40 +/- 31 to 82 +/- 49 (power loss). These calculations suggest that both changing viscosity and a changing value for the junction exponent are likely important when examining the energy optimization within the arteriolar microcirculation.

L-arginine-induced conducted signals alter upstream arteriolar responsivity to L-arginine.

Our purpose was to determine whether L-arginine was involved in vascular communication between downstream and upstream locations within a defined microvascular region. Arteriolar diameter was measured for the branches along a transverse arteriole in the superfused cremaster of anesthetized (pentobarbital sodium, 70 mg/kg i.p.) hamsters (N = 53). The upstream branch arterioles dilated significantly to locally applied L-arginine (100 mumol/L pipette concentration) only if the downstream branches (approximately 1400 microns away) were preexposed. With exposure order downstream to upstream, diameter change was last branch, -3.8 +/- 1.5% (of baseline); third, +58.1 +/- 27%; first, +92 +/- 26% (n = 5); with exposure order upstream to downstream: first branch, -0.4 +/- 3%; third, +5 +/- 11%; last, -5.6 +/- 7.5% (n = 4). Thus, downstream preexposure to L-arginine altered the responsivity upstream to locally applied L-arginine. Downstream-applied L-arginine also induced a conducted vasodilation (+17.8 +/- 2.8%; n = 14) 1327 +/- 166 microns upstream. This response was completely blocked by simultaneous sucrose (600 mOsm), halothane (0.0345%), or N omega-nitro-L-arginine (L-NNA, 100 mumol/L) exposure to the feed vessel (second micropipette) midway between the downstream site of L-arginine exposure and the upstream observation site. An acetylcholine-induced conducted vasodilation (+18.1 +/- 2.6%, n = 8) was also completely blocked by sucrose, halothane, or L-NNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Arteriolar bifurcation angles vary with position and when flow is changed.

Flow distribution at microvascular bifurcations is influenced by the geometry of the bifurcation region, an area which also is altered pathologically. Bifurcation geometry was measured by in vivo microscopy of the cremaster muscle of anesthetized (Nembutal, 70 mg/kg) golden hamsters (N = 40), at rest and during maximal dilation (10(-4) M adenosine). The sequential branches had progressively smaller angles of bifurcation at rest: (first position) 118 +/- 5 degrees; (second) 89 +/- 6 degrees; (third) 78 +/- 5 degrees; (last) 58 +/- 4 degrees. Between flow conditions, the angle at any bifurcation changed by up to +/- 50 degrees, and the angle change was related to position. For the first position, the angles that decreased vs those that increased were significantly different at rest (130 +/- 6 degrees vs 109 +/- 7 degrees), but not during maximal dilation (119 +/- 6 degrees vs 118 +/- 7 degrees). Conversely, at the last bifurcation, the resting angles were not different (58 +/- 5 degrees vs 56 +/- 7 degrees), but became significantly different during maximal dilation (48 +/- 6 degrees vs 68 +/- 6 degrees). The axial distance to the first branch ranged between 57 and 857 microns; the angle at the first position was significantly smaller for those first branches that arose further distally along the feed. Further, angles that decreased (vs those that increased) were from significantly longer transverse arterioles (total length: 1820 +/- 77 microns vs 1560 +/- 61 microns). Resting tone was related to the angle as the smaller angles at the first position, but not at the last position, were more constricted in diameter. Tone was differently related to angle change as the bifurcations that decreased (vs those that increased) in angle were significantly more constricted at the last position (branch diameter rest/maximal: 0.57 +/- 0.05 vs 0.81 +/- 0.08) but not at the first position (0.66 +/- 0.09 vs 0.64 +/- 0.05). Thus, we show that the angle of bifurcation varies systematically for sequential branches arising along a single transverse arteriole and that the angles change with flow. This systematic organization for the geometric shape of sequential bifurcation regions may participate in the regulation of flow distribution within this group of arterioles.

Regulation of capillary perfusion by small arterioles is spatially organized.

To explore a mechanism for spatial recruitment of capillaries, this study determined whether the arterioles controlling capillary perfusion, which typically arise as sequential branches along a transverse arteriole, could respond differently from each other in situ in a spatially ordered way. Diameter changes were measured for these arterioles at a known location in the intact microvasculature in the cremaster muscle of anesthetized Golden hamsters (N = 67); each arteriole controls separate capillary groups. These arterioles all had the same concentration dependence to locally (by micropipette) applied norepinephrine (NE, 10(-9) to 10(-3) mol/L), and 10(-9) mol/L NE did not induce diameter changes when applied locally to individual vessels. However, 10(-9) mol/L NE added to the tissue superfusate, or 5% added superfusate oxygen (also locally subthreshold), each induced significant diameter changes (both constrictions and dilations), in different branches, that were presumably due to summation of individually subthreshold events that changed the prevailing conditions at the point of observation. These significant diameter changes were related to the maximal diameter or to initial tone of the branches, but these changes occurred in different ways for NE versus oxygen. With NE, the branch arterioles that constricted (versus dilated) were significantly larger (maximal diameter, 22.3 +/- 2.6 versus 15.9 +/- 2.1 microns) and had higher tone (fractional constriction, 0.53 +/- 0.05 versus 0.63 +/- 0.05); with oxygen, those that constricted were the same size as those that dilated (maximal diameter, 28.6 +/- 1.1 versus 30.5 +/- 2.7 microns), but constrictors had lower tone (fractional constriction, 0.49 +/- 0.04 versus 0.39 +/- 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Mn and Cd transport by the Na-Ca exchanger of ferret red blood cells.

Ca influx via the Na-Ca exchanger into ferret red blood cells is easily measured from a Na-free solution; the intracellular Na concentration is normally approximately 150 mM in ferret red blood cells. We have found that Mn and Cd competitively inhibit Ca influx. Mn influx and Cd influx were a saturable function of the divalent cation concentration, consistent with a carrier mechanism. Indeed, the Km (approximately 10 microM) and the Vmax (usually 1-3 mmol.l packed cells-1.h-1) were similar for Ca, Cd, and Mn. Extracellular Na inhibited divalent cation influx, and intracellular Na stimulated influx. These results are consistent with Na-Cd and Na-Mn influx pathways in ferret red blood cells. Ca (1 mM) almost completely inhibited Mn influx and Cd influx, whereas 1 mM Mg inhibited 5-15%. These results strongly support the notion that Mn and Cd are alternative substrates for Ca on the ferret red cell Na-Ca exchanger. The similarity in the behavior of all three divalent cation places important constraints on kinetic and structural models of the exchanger.

Kinetic models of Na-Ca exchange in ferret red blood cells. Interaction of intracellular Na, extracellular Ca, Cd, and Mn.

The kinetic equation that best describes the intracellular Na dependence of Ca influx into ferret red cells is sequential; whether this implies that there is a conformation of the protein that has both Na and Ca ions bound remains to be determined. Cd and Mn substitute very well for Ca on the exchanger in ferret red cells; this suggests that the Ca-binding site does not contain an important thiol and that the one of the Na steps may be rate limiting.