Simvastatin and GGTI-2133, a geranylgeranyl transferase inhibitor, increase erythrocyte deformability but reduce low O(2) tension-induced ATP release.

Statin drugs inhibit 3-hydroxy-3-methylglutaryl CoA reductase, which reduces the synthesis of both cholesterol and isoprenoids (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), with the latter being lipid molecules responsible for the posttranslational modification of small GTP-binding proteins such as Rho. Effects of statins, independent of lowering blood cholesterol levels, are thought to occur by inhibition of Rho/Rho kinase. The Rho kinase inhibitor Y-27632 has been reported to increase both erythrocyte deformability and low O2 tension-induced ATP release. Here, we tested the hypothesis that by inhibiting Rho/Rho kinase, simvastatin would increase both erythrocyte deformability and low O2 tension-induced ATP release. Male Sprague-Dawley rats were divided into two groups, control or simvastatin treated [simvastatin-supplemented chow (0.02%)], for 4 wk. Simvastatin treatment increased rat erythrocyte deformability compared with controls (n = 6, P < 0.05). However, erythrocytes of simvastatin-treated rats (n = 9, P < 0.05) exhibited impaired low O2 tension-induced ATP release. Similarly, the geranylgeranyl transferase inhibitor GGTI-2133 (10 µM) also increased deformability and impaired low O2 tension-induced ATP release in healthy human erythrocytes (P < 0.05). Interestingly, ATP release in response to mastoparan 7 (n = 7, P < 0.05), which directly activates Gi, and isoproterenol (n = 5, P < 0.05), which signals through Gs, was not altered by incubation with GGTI-2133. These results suggest that although statins increase erythrocyte deformability, likely by inhibiting geranylgeranylation, the finding that both statins and a geranylgeranyl transferase inhibitor attenuated low O2 tension-induced ATP release demonstrates that factors in addition to erythrocyte deformability are critical for ATP release in response to this physiological stimulus.

The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release.

Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 µM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 µM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.

Role of erythrocyte-released ATP in the regulation of microvascular oxygen supply in skeletal muscle.

In a 1914 book entitled The Respiratory Function of the Blood, Joseph Barcroft stated that 'the cell takes what it needs and leaves the rest'. He postulated that there must be both a 'call for oxygen' and a 'mechanism by which the call elicits a response...' In the past century, intensive investigation has provided significant insights into the haemodynamic and biophysical mechanisms involved in supplying oxygen to skeletal muscle. However, the identification of the mechanism by which tissue oxygen needs are sensed and the affector responsible for altering the upstream vasculature to enable the need to be appropriately met has been a challenge. In 1995, Ellsworth et al. proposed that the oxygen-carrying erythrocyte, by virtue of its capacity to release the vasoactive mediator ATP in response to a decrease in oxygen saturation, could serve both roles. Several in vitro and in situ studies have established that exposure of erythrocytes to reduced oxygen tension induces the release of ATP which does result in a conducted arteriolar vasodilation with a sufficiently rapid time course to make the mechanism physiologically relevant. The components of the signalling pathway for the controlled release of ATP from erythrocytes in response to exposure to low oxygen tension have been determined. In addition, the implications of defective ATP release on human pathological conditions have been explored. This review provides a perspective on oxygen supply and the role that such a mechanism plays in meeting the oxygen needs of skeletal muscle.

Capillary oxygen transport during severe hypoxia: role of hemoglobin oxygen affinity.

The efficacy of an increased hemoglobin oxygen affinity [decreased oxygen half-saturation pressure of hemoglobin (P50)] on capillary oxygen transport was evaluated in the hamster retractor muscle under conditions of a severely limited oxygen supply resulting from the combined effects of a 40% reduction in systemic hematocrit and hypoxic ventilation (inspired oxygen fraction 0.1). Two groups of hamsters were utilized: one with a normal oxygen affinity (untreated; P50 = 26.1 +/- 2.4 Torr) and one with an increased oxygen affinity (treated; P50 = 15.7 +/- 1.4 Torr) induced by the chronic short-term administration of sodium cyanate. Using in vivo video microscopy and image analysis techniques, we determined oxygen saturation and associated hemodynamics at both ends of the capillary network. During hypoxic ventilation, the decrease in oxygen saturation across the network was 3.6% for untreated animals compared with 9.9% for treated animals. During hypoxia, estimated end-capillary PO2 was significantly higher in the untreated animals. These data indicate that, at the capillary level, a decreased P50 is advantageous for tissue oxygenation when oxygen supply is severely compromised, because normal oxygen losses in capillaries are maintained in treated but not in untreated animals. The data are consistent with the presence of a diffusion limitation for oxygen during severe hypoxia in animals with a normal hemoglobin oxygen affinity.

Impaired release of ATP from red blood cells of humans with primary pulmonary hypertension.

Previously, we reported that in the isolated perfused rabbit lung, red blood cells (RBCs) obtained from either rabbits or healthy humans were a required component of the perfusate to unmask evidence of nitric oxide (NO) participation in regulation of the pulmonary circulation. In addition, we found that mechanical deformation of rabbit and healthy human RBCs released ATP, a known agonist for enhanced NO synthesis. In contrast, RBCs obtained from patients with cystic fibrosis (CF) did not release ATP in response to mechanical deformation. The coexistence of airway disease and alveolar hypoxia in patients with CF precluded the drawing of conclusions relating a defect in RBC ATP release with the pulmonary hypertension associated with CF. Airway disease and alveolar hypoxia are not, however, features of primary pulmonary hypertension (PPH), a human condition of unknown etiology. We postulated that a defect in NO generation might contribute to the increased pulmonary vascular resistance in PPH, and as a first step, we hypothesized that RBCs obtained from patients with PPH would not release ATP. In contrast to RBCs of healthy humans, when RBCs of PPH patients were passed through filters (average pore size 12, 8, or 5 microm), ATP was not released and the RBCs exhibited reduced deformability. Moreover, when incubated with the active cAMP analogue, Sp-cAMP (100 microM), an activator of the CF transmembrane conductance regulator, ATP was not released. These results demonstrate that RBCs obtained from patients with PPH fail to release ATP whether the stimulus is mechanical or pharmacological. Thus, failure of RBCs to release ATP in patients with PPH might be a major pathogenetic factor that accounts for the heretofore unknown etiology of their pulmonary hypertension.

Erythrocytes as controllers of perfusion distribution in the microvasculature of skeletal muscle.

In 1929, August Krogh identified the matching of oxygen (O(2)) supply with demand in skeletal muscle as a fundamental physiological process. In the intervening decades, much research has been focused on elucidating the mechanisms by which this important process occurs. For any control system to be effective, there must be a means by which the need is determined and a mechanism by which that information is coupled to an appropriate response. The focus of this review was to highlight current research in support of the hypothesis that the mobile erythrocyte, when exposed to reduced O(2) tension, releases ATP in a controlled manner. This ATP interacts with purinergic receptors on the endothelium producing both local and conducted vasodilation enabling the erythrocyte to distribute perfusion to precisely match O(2) delivery with need in skeletal muscle. If this is an important mechanism for normal physiological control of microvascular perfusion, defects in this process would be anticipated to have pathophysiological consequences. Individuals with either type 2 diabetes (DM2) or pre-diabetes have microvascular dysfunction that contributes to morbidity and mortality. DM2 erythrocytes and erythrocytes incubated with insulin at levels similar to those seen in pre-diabetes fail to release ATP in response to reduced O(2) tension. Knowledge of the components of the signal transduction pathway for low O(2) -induced ATP release suggest novel therapeutic approaches to ameliorating this defect. Although the erythrocyte may be but one component of the complex O(2) delivery process, it appears to play an important role in distributing oxygen within the microvasculature.

The red blood cell as an oxygen sensor: what is the evidence?

The matching of oxygen supply with demand requires the existence of a mechanism within the tissue capable of both sensing tissue oxygen need and inducing alterations in vascular perfusion necessary to meet that need. Historically, localized sites within the tissue and the vessels themselves have been investigated with the sensor site failing to be determined. Within the last decade, studies have focused on the red blood cell, the efficient carrier of oxygen, as a possible candidate. The red blood cell is clearly capable of sensing oxygen levels, as its extent of haemoglobin desaturation (decrease in oxygen content) is intimately tied with tissue oxygen demand. In addition, numerous studies have indicated that the red blood cell is capable of releasing increased amounts of adenosine 5'-triphosphate (ATP) as oxygen content falls and its haemoglobin becomes desaturated. Within the vasculature, intraluminal ATP has been shown to induce a vasodilator response which is conducted along the vessels resulting in augmentation of tissue perfusion. While details of the red blood cell's role are still under investigation, evidence presented here supports the intriguing idea that the mobile red blood cell may itself be able to augment blood flow and oxygen delivery wherever and whenever the need might arise. Such a mechanism eliminates the requirement for a diverse network of sensing sites throughout the vasculature and should provide a more efficient means of appropriately matching oxygen supply with demand.

Heterogeneity of oxygen diffusion through hamster striated muscles.

We determined the O2 diffusion coefficients (DO2) and resting O2 consumptions for three hamster muscles with differing histochemical fiber type composition using a nonsteady-state technique. The muscles ranged from an oxidative muscle (soleus) to a glycolytic muscle (sartorius) with a third, the cheek pouch retractor, having mixed metabolic properties. We found that the DO2 of the soleus (corrected to 37 degrees C) was 2.59 +/- 0.33 X 10(-5) cm2/s, whereas the DO2 of the sartorius was 1.15 +/- 0.14 X 10(-5). The value for the retractor was between these two values (1.39 +/- 0.14 X 10(-5) cm2/s). In addition, we observed a linear correlation between DO2 and the percent of transverse cross-sectional area occupied by oxidative fibers, suggesting that the differences in DO2 can be accounted for by differences in their histochemical fiber type composition. In light of known differences in capillary density and maximal O2 demand, these data imply an interplay between capillarity and O2 diffusion rate resulting in an appropriate O2 delivery to meet each muscle's maximal O2 demand.

Microvascular oxygen transport: impact of a left-shifted dissociation curve.

The impact of an increased hemoglobin oxygen affinity (decreased P50) on oxygen transport was evaluated in capillaries of the retractor muscle under nonhypoxic (FIo2 = 0.30 and 0.21) and hypoxic (FIo2 = 0.10) conditions in hamsters with normal oxygen affinity [control; P50 = 26.1 +/- 1.0 (SD) mmHg, n = 12] and in hamsters with an increased oxygen affinity [treated; P50 = 16.2 +/- 1.6 (SD) mmHg, n = 7] induced by chronic short-term administration of sodium cyanate. Using in vivo video microscopy and computer-aided image analysis, we determined oxygen saturation (SO2) and associated hemodynamic parameters in both arteriolar (n = 30 control, 18 treated) and venular (n = 25 control, 17 treated) capillaries. In response to hypoxia, systemic arterial PO2 decreased to 29.6 +/- 6.0 (SD) mmHg in control animals and 24.7 +/- 3.8 (SD) mmHg in treated animals associated with abrupt decreases in systemic arterial blood pressure and increases in respiratory rate. The decrease in SO2 across the capillary network during nonhypoxic ventilation was 13.3% SO2 for control animals and 11.0% SO2 for treated animals. During hypoxic ventilation, the decrease in SO2 was 9.1% SO2 in control animals and 8.7% SO2 in treated animals. Hemodynamic parameters were not significantly different in the two groups during hypoxia. Estimated end-capillary PO2 was significantly lower in the treated animals. These data indicate that an increased oxygen affinity does not provide an obvious advantage for oxygen transport during hypoxia at the level of the capillary network in resting striated muscle; however, such an advantage might become apparent in the presence of an increased metabolic rate or a more severe hypoxic challenge.

Arterioles supply oxygen to capillaries by diffusion as well as by convection.

In the early part of this century, August Krogh proposed a model of oxygen transport in capillaries that assumes that all oxygen is delivered to the capillaries by convection from small terminal arterioles and lost from these capillaries by diffusion. This model and its consequences have been used extensively to interpret whole organ oxygen transport data in terms of diffusion between capillaries and tissues and to relate changes in microvascular hemodynamics to alterations in oxygen transport. We evaluated the appropriateness of such extrapolation by measuring oxygen saturation at discrete locations along the lengths of individual capillaries in the hamster cheek pouch retractor muscle. Our results indicate that the amount of oxygen lost from individual capillaries can be markedly affected by the presence of larger microvessels that frequently cross the capillary path. These larger vessels act either as a diffusive supply of oxygen for the red blood cells within the capillary or as an additional sink for the oxygen depending on the direction of the oxygen tension gradient. This transfer of oxygen between larger microvessels and capillaries attenuates the importance of capillary hemodynamics in oxygen exchange. Therefore, conclusions about local oxygen exchange that utilize only hemodynamic data from whole organ or microvascular experiments and the Krogh model will generally be invalid and should be viewed with caution.

Estimation of red cell flow microvessels: consequences of the Baker-Wayland spatial averaging model.

The dual sensor cross-correlation method of H. Wayland and P.C. Johnson [1967), J. Appl. Physiol. 22, 333-337) has become a standard technique for determining the velocity of red blood cells (RBCs) in glass tubes and blood vessels. M. Baker and H. Wayland [1974), Microvasc. Res. 7, 131-143) found that under a variety of conditions the ratio of dual sensor velocity at the centerline of a glass tube to the blood velocity averaged over the lumen was close to 1.6. They provided an explanation of this factor based on spatial averaging of RBC velocity vertically through the tube as well as laterally across the face of the sensor. Their spatial averaging model could also account for the apparent blunting of RBC velocity profiles determined with the dual sensor technique. We used Baker and Wayland's spatial averaging model to calculate how the above velocity ratio depends on sensor size. A nonlinear relation between the velocity ratio and sensor size was found such that the velocity ratio varied from 1.6 to 1.33 as the ratio of sensor width to vessel or tube diameter was varied from 0 to 1. These results also hold for vessels or tubes of elliptic cross section. Some investigators have found that the velocity of red cells near the walls of blood vessels can be a substantial fraction of centerline velocity which suggests that RBC velocity distributions can be blunter than a Poiseuille distribution. We repeated the above calculation for blunted parabolic profiles and we found that the velocity ratio ranged from 1 for plug flow to 1.6 for Poiseuille flow. These calculations show that reliable estimates of RBC flow from dual sensor centerline velocity measurements require one to take into account the relative size of the sensor and blood vessel diameter as well as the bluntness of the RBC velocity distribution.

Evaluation of photometric methods for quantifying convective mass transport in microvessels.

We evaluated the accuracy of blood (Qb), hemoglobin (QHb) and O2 (QO2) flow determinations in arterioles (30-89 micron ID) and venules (30-140 micron ID) of the hamster retractor muscle by testing flow conservation in straight segments (for Qb and QHb) and at bifurcations (for Qb, QHb, and QO2). We found absolute values of relative discrepancies in flow conservation of greater than 30% in straight sections and of greater than 20% at bifurcations. To understand these results, we evaluated the assumptions implicit in the flow calculations by determining the intraluminal profiles of red blood cell velocity, hemoglobin concentration, and O2 saturation. We found that these assumptions were not generally satisfied since only 59% of velocity profiles, 70% of hemoglobin concentration profiles, and 70% of O2 saturation profiles were symmetric about the vessel axis with the velocity profiles more blunt than would be expected for Poiseuille flow. We reevaluated flow conservation by taking into account the bluntness of the velocity profiles. By selecting vessels that had symmetric velocity and hemoglobin concentration profiles and by including the bluntness of the velocity profile, we obtained a significant improvement in the demonstration of flow conservation.

Hypoxic pulmonary vasoconstriction during steady and pulsatile flow in ferrets.

We examined the effects of hypoxia and pulsatile flow on the pressure-flow relationships in the isolated perfused lungs of Fitch ferrets. When perfused by autologous blood from a pump providing a steady flow of 60 ml/min, the mean pulmonary arterial pressure rose from 14.6 to 31.3 Torr when alveolar PO2 was reduced from 122 to 46 Torr. This hypoxic pressor response was characterized by a 10.1-Torr increase in the pressure-axis intercept of the extrapolated pressure-flow curves and an increase in the slope of these curves from 130 to 240 Torr X l-1 X min. With pulsatile perfusion from a piston-type pump, mean pulmonary arterial pressure increased from 17.5 to 36.3 Torr at the same mean flow. This hypoxic pressor response was also characterized by increases in the intercept pressure and slope of the pressure-flow curves. When airway pressure was raised during hypoxia, the intercept pressure increased further to 25 +/- 1 Torr with a further increase in vascular resistance to 360 Torr X l-1 X min. Thus, in contrast to the dog lung, in the ferret lung pulsatile perfusion does not result in lower perfusion pressures during hypoxia when compared with similar mean levels of steady flow. Since the effects of high airway pressure and hypoxia are additive, they appear to act at or near the same site in elevating perfusion pressure.

Assessment and impact of heterogeneities of convective oxygen transport parameters in capillaries of striated muscle: experimental and theoretical.

Convective oxygen transport parameters were determined in arteriolar (n = 5) and venular (n = 5) capillary networks in the hamster cheek pouch retractor muscle. Simultaneously determined values of red blood cell velocity, lineal density, red blood cell frequency, hemoglobin oxygen saturation (SO2), oxygen flow (QO2), longitudinal SO2 gradient, and diameter were obtained in a total of 73 capillaries, 39 at the arteriolar ends of the network (arteriolar capillaries) and 34 at the venular ends (venular capillaries). We found that the hemodynamic variables were not different at the two ends. However, not unexpectedly, SO2 and QO2 were significantly higher at the upstream end of arteriolar capillaries (60.8 +/- 9.8 (SD)% and 0.150 +/- 0.081 pl/sec, respectively) compared with the downstream end of venular capillaries (39.9 +/- 13.6% and 0.108 +/- 0.095 pl/sec, respectively). Heterogeneities in red blood cell velocity, lineal density, SO2, and QO2, assessed by their coefficients of variation, were significantly greater in venular capillaries. To evaluate the impact of these heterogeneities on oxygen exchange, we incorporated these unique experimental data into a mathematical model of oxygen transport which accounts for variability in red blood cell frequency, lineal density, inlet SO2, capillary diameter, and, to some degree, capillary flow path lengths. An unexpected result of the simulation is that only the incorporation of variability in capillary flow path lengths had any marked effect on the heterogeneity in end-capillary SO2 in resting muscle due to extensive diffusional shunting of oxygen among adjacent capillaries. We subsequently evaluated, through model simulations, the effect of these heterogeneities under conditions of increased flow and high oxygen consumption. Under these conditions, the model predicts that heterogeneities in the hemodynamic parameters will have a marked effect on oxygen transport in this muscle.

Relationship between capillary and systemic venous PO2 during nonhypoxic and hypoxic ventilation.

We evaluated the relationship between end-capillary and systemic venous PO2 values in the retractor muscle of 14 anesthetized hamsters during both nonhypoxic and hypoxic ventilation to ascertain whether the level of tissue oxygenation could be reliably estimated from the systemic parameter. End-capillary PO2 was estimated from measurements of oxygen saturation in capillaries at the venular end of the network obtained using in vivo video microscopy and computer-aided image-analysis techniques at three different levels of inspired oxygen (0.3, 0.21, and 0.1). Measurements of systemic arterial and venous blood gases were made in conjunction with these capillary determinations. In addition, in a portion of the study we utilized an oxygen microelectrode to determine the PO2 in the first-order venule draining the portion of the muscle containing the capillaries under study. We found that only when the animals were made acutely hypoxic was there any correspondence between the systemic venous and end-capillary PO2 values. In addition, these data provide support for the presence of arteriovenous shunting of oxygen during nonhypoxic ventilation.

Arteriolar responses to extracellular ATP in striated muscle.

Blood flow and its distribution must be appropriately regulated to ensure that perfusion is matched to local tissue demands. We investigated the role of ATP in triggering a conducted alteration in arteriolar diameter in the Saran-covered cheek pouch retractor muscle of anesthetized hamsters (n = 60). Vascular responses were observed using in vivo video microscopy upstream from the site of micropressure application of ATP (10(-8)-10(-4) M) either into the lumen or just outside the wall of first- and second-order arterioles. The role of nitric oxide (NO) in the vascular responses to ATP was determined by inhibiting NO synthase activity with N(omega)-nitro-L-arginine methyl ester (L-NAME) with and without coadministration of an excess of L-arginine. Intraluminal application of ATP led to a concentration-dependent vasodilation, which was conducted upstream along the arteriole. The dilatory response was blocked by systemic pretreatment with L-NAME and was maintained in the presence of an excess of L-arginine. In contrast, ATP introduced extraluminally resulted in a conducted vasoconstrictor response that was enhanced by pretreatment with L-NAME. The dilator response to intraluminal ATP, in the context of ATP release from erythrocytes under conditions associated with decreased supply relative to demand, supports a role for the erythrocyte in communicating local tissue needs to the vasculature, enabling the appropriate matching of oxygen supply to demand.

The pineal body of the dog.

A technique is described for finding the pineal body of the dog. The posterior half of the skull is cut a little behind the parietofrontal suture, through the occipital condyles. The cerebral hemispheres and cerebellum are carefully sliced, disclosing the pineal at the frontal edge of the colliculi. Two types of cells are present, those with completely round nuclei, and others with vesicular and variably shaped nuclei. In the histological pattern, ependymal cells were observed on the edges, pinealocytes and glial cells within the gland.