Direct effects of altered temperature on renal structure and function

Although marked alterations in temperature often accompany ischemic, acute renal failure (ARF), the effects of altered temperature on renal structure and function have received little attention. In the present investigation, isolated rat kidneys perfused at 41 degrees C had extensive tubular damage and decreased function compared to kidneys perfused at 37 degrees C. In contrast, kidneys perfused at 30 degrees C had less tubular damage, and better function, than kidneys perfused at 37 degrees C. Increased temperature caused a 50% reduction in renal ATP (0.46 +/- 0.04 microM/100 mg tissue protein. 37 degrees C vs. 0.26 +/- 0.03 microM/100 mg tissue protein, 41 degrees C; p less than 0.05). The decreased ATP occurred despite reduced sodium reabsorption (129 +/- 8 microM/min/g, 37 degrees C vs. 65 +/- 12 microM/min/g, 41 degrees C, p less than 0.05) and normal renal oxygen consumption (QO2). These results suggest that increased temperature may cause an uncoupling of QO2 and sodium chloride transport, and an increase in nontransport mediated, basal metabolic rate may result in depleted cellular ATP levels and renal tubular cell death.     

Functional and morphological studies of protein transcytosis in continuous endothelia

Continuous microvascular endothelium constitutively transfers protein from vessel lumen to interstitial space. Compelling recent biochemical, ultrastructural, and physiological evidence reviewed herein demonstrates that protein transport is not the result of barrier "leakiness" but, rather, is an active process occurring primarily in a transendothelial vesicular pathway. Protein accesses the vesicular pathway by means of caveolae open to the vessel lumen. Vascular tracer proteins appear in free cytoplasmic vesicles within minutes; contents of transport vesicles are rapidly deposited into the subendothelial matrix by exocytosis. Caveolin-1 deficiency eliminates caveolae and abolishes vesicular protein transport; interestingly, exchange vessels develop a compensatory transport mode through the opening of a paracellular permeability pathway. The evidence supports the transcytosis hypothesis and the concept that transcytosis is a fundamental component of transendothelial permeability of macromolecules.     

Transendothelial transport (transcytosis) of iron-transferrin complex in the rat liver

To determine the transport pathway of iron-transferrin complex (Fe-TF) into the hepatocyte, we labeled Fe-TF with colloidal gold and perfused rat liver through the portal vein with this probe under different conditions. The tissue was then studied by transmission electron microscopy. At a cold temperature (approximately 4 degrees C), the probe bound to the luminal surface of sinusoidal endothelium without internalization. The binding was limited to the endothelium and there was no binding to Kupffer cells or hepatocytes. The binding was inhibitable in the presence of excess soluble Fe-TF, indicating the specificity of the bindings. At 37 degrees C the probe was internalized via a system of coated pits and vesicles. Morphometric analyses of the temporal sequence of events suggested that the probe was transported, in part, across the endothelium via a system of tubules and vesicles and externalized on the abluminal side via a system of coated pits. The probe was then taken up by hepatocytes. Only minimal uptake was noted when gold-labeled bovine serum albumin was used either at the low temperature or at 37 degrees C. The findings suggest that the liver uptake of Fe-TF complex by hepatocytes is a transendothelial phenomenon (transcytosis).     

Ultrastructural and hormonal metabolic studies of rat liver maintained in vitro by perfusion at 30 degrees C and 37 degrees C: a time course study by TEM, SEM and RIA

Isolated rat liver perfusion system has been extensively used for metabolic and functional studies. Results derived from the application of this system may reflect true biochemical changes but they may also be associated with some structural changes. This study was undertaken to correlate the cytological changes and functional integrity of isolated rat liver perfused in vitro at normal physiological temperature (37 degrees C) and 30 degrees C, using a non-recirculating system. The livers were perfused for 3 hours with modified Ham's F10 culture medium supplemented with thyroxine hormone (T4). The hepatocyte structural integrity was studied by light microscopy, transmission and scanning electron microscopy. The triiodothyronine (T3) and T4 hormones in the perfusion medium and the effluent fractions were assessed by radioimmunoassay. The livers perfused at 30 degrees C remained morphologically intact at the ultrastructural level for 3 hours whilst at 37 degrees C, hepatocytes in the centrilobular zone exhibited marked structural alterations. The percentage of T4 uptake was significantly higher (P less than 0.01) in livers perfused at 30 degrees C (50.8 +/- 7.7% vs 38 +/- 7.7%, 37 degrees C), but the net T3 output (3.16 +/- 1.04 micrograms) and the conversion of T4 to T3 (4 +/- 0.62%) were significantly higher (P less than 0.001) in livers perfused at 37 degrees C in comparison to livers perfused at 30 degrees C (1.61 +/- 0.84 micrograms and 1.68 +/- 0.76%, respectively). In conclusion, at 30 degrees C the hepatic T4 uptake is not inhibited, but the rate of T4 to T3 conversion has decreased, additionally the livers remain morphologically well preserved throughout the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a second valve system in lymphatics: endothelial microvalves.

The mechanism for interstitial fluid uptake into the lymphatics remains speculative and unresolved. A system of intralymphatic valves exists that prevents reflow along the length of the lymphatic channels. However, these valves are not sufficient to provide unidirectional flow at the level of the initial lymphatics. We investigate here the hypothesis that initial lymphatics have a second, separate valve system that permits fluid to enter from the interstitium into the initial lymph channels but prevents escape back out into the tissue. The transport of fluorescent microspheres (0.31 microm) across endothelium of initial lymphatics in rat cremaster muscle was investigated with micropipette manipulation techniques. The results indicate that microspheres can readily pass from the interstitium across the endothelium into the lumen of the initial lymphatics. Once inside the lymphatic lumen, the microspheres cannot be forced out of the lumen even after elevation of the lymphatic pressure by outflow obstruction. Reaspiration of the microspheres inside the lymphatic lumen with a micropipette is blocked by the lymphatic endothelium. This blockade exists whether the aspiration is carried out at the microsphere entry site or anywhere along the initial lymphatics. Nevertheless, puncture of the initial lymphatic endothelium with the micropipette leads to rapid aspiration of intralymphatic microspheres. Investigation of lymphatic endothelial sections fixed during lymph pumping shows open interendothelial junctions not found in resting initial lymphatics. These results suggest that initial lymphatics have a (primary) valve system at the level of the endothelium. In conjunction with the classical (secondary) intralymphatic valves, the primary valves provide the mechanism that facilitates the unidirectional flow during periodic compression and expansion of initial lymphatics.     

Increased capillary permeability in muscularis layer of rat intestine caused by kidney extract.

Kidney extract and synthetic angiotensin II were injected into bilaterally nephrectomized rats in dosages capable of raising the mean arterial pressure by about 20 mmHg. Changes in ultrastructure and permeability for ferritin molecules were then examined in capillaries located in muscularis layer of the intestinal walls. Kidney extract with a high renin content was obtained from the renal cortex of rats by means of stepwise centrifugation methods. Animals injected with saline served as controls. In rats receiving kidney extract tissue edema was observed in the spaces around the blood and lymphatic capillaries. In these spaces ferritin molecules accumulated in high concentration indicating plasma protein leakage. Ferritin molecules within the endothelium were restricted within plasmalemmal vesicles, but were not found within interendothelial junctions or within the cytoplasmic matrix. Morphometric analysis of vesicular transport in the endothelial cells revealed a significant increase in labeling rate for the vesicles with ferritin molecules. These results suggest that the kidney extract contains substance(s) which increase capillary permeability for plasma proteins at least via increased vesicular transport, resulting in tissue edema.     

Low cytoplasmic pH inhibits endocytosis and transport from the trans- Golgi network to the cell surface

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.     

The renal cortical lymphatic system in the rat, hamster, and rabbit

Rat, hamster, and rabbit renal cortical lymphatics were examined by light and electron microscopy. Rat and hamster kidneys possessed both intra- and interlobular lymphatics that were structurally similar at the light microscopic level. Ultrastructural examination of the hamster lymphatic endothelium, however, revealed an unusual arrangement of cytoplasmic extensions not seen in the other two species. The intralobular lymphatics were related primarily to tubules, afferent arterioles, and renal corpuscles and were consistent with lymph formation from both plasma filtrate and tubular reabsorbate. Interlobular lymphatics were seen in connective tissue associated with the interlobular blood vessels. Rabbit cortex contained only interlobular lymphatics. Cross-sectional area, maximum diameter, volume density, and profile density were determined by stereological measurements using a computer-based image analyzer. The morphological data from the rat were used, in combination with published values for lymph flow, to calculate the rate of lymph formation per unit area of endothelium in lymphatics of the renal cortex. Among kidneys fixed by retrograde perfusion, the cortical lymphatic system was most extensive in maximum diameter, volume density, and profile density. It was smallest in the rabbit and intermediate in the rat. Lower volume and profile density were found for rat kidneys fixed by the dripping technique. It was concluded that: tubular reabsorbate probably contributes to renal lymph in the rat and hamster, but not in the rabbit; significant differences exist in the extent of the renal lymphatic systems among the three species, with the hamster kidney having the richest network and the rabbit the poorest; the method of fixation influences the measured size and density of renal cortical lymphatics; and the estimated rate of lymph formation in the kidney of the rat is roughly comparable to that in the dog.     

Discontinuous LYVE-1 expression in corneal limbal lymphatics: dual function as microvalves and immunological hot spots

LYVE-1(+) corneal lymphatics contribute to drainage and immunity. LYVE-1 is widely accepted as the most reliable lymphatic marker because of its continuous expression in lymphatic endothelium. LYVE-1 expression in corneal lymphatics has not been examined. In this study, we report intact CD31(+) corneal lymphatic capillary endothelial cells that do not express LYVE-1. The number of LYVE-1(-) gaps initially increased until 8 wk of age but was significantly reduced in aged mice. C57BL/6 mice showed a notably higher number of the LYVE-1(-)/CD31(+) lymphatic regions than BALB/c mice, which suggests a genetic predisposition for this histological feature. The LYVE-1(-) lymphatic gaps expressed podoplanin and VE-cadherin but not αSMA or FOXC2. Interestingly, the number of LYVE-1(-) gaps in FGF-2, but not VEGF-A, implanted corneas was significantly lower than in untreated corneas. Over 70% of the CD45(+) leukocytes were found in the proximity of the LYVE-1(-) gaps. Using a novel in vivo imaging technique for visualization of leukocyte migration into and out of corneal stroma, we showed reentry of extravasated leukocytes from angiogenic vessels into newly grown corneal lymphatics. This process was inhibited by VE-cadherin blockade. To date, existence of lymphatic valves in cornea is unknown. Electron microscopy showed overlapping lymphatic endothelial ends, reminiscent of microvalves in corneal lymphatics. This work introduces a novel corneal endothelial lymphatic phenotype that lacks LYVE-1. LYVE-1(-) lymphatic endothelium could serve as microvalves, supporting unidirectional flow, as well as immunological hot spots that facilitate reentry of stromal macropahges.     

Peroxidase transport through the endotheliocytes of the microvasculature of the rat jejunal villi

The use of horseradish peroxidase as a macromolecular tracer showed partial differences in paracellular and transcellular pathways of the net transendothelial protein transport related to the segmental belonging of microvessels. The most likely pathway of transport through precapillary arteriolar endothelium is poorly expressed vesicular transfer. High endothelial permeability in subepithelial and marginal capillary and postcapillary venule of intestinal villi for horseradish peroxidase, is mostly due to vesicular transport and open junctions. Transendothelial channels and fenestrations in exchange microvessel endothelius were marked by horseradish peroxidase.     

Transendothelial transport (transcytosis) of iron-transferrin complex in the bone marrow

To determine the transport pathway of iron-transferrin complex (Fe-TF) across the marrow-blood barrier, we labeled Fe-TF with colloidal gold and perfused rat femoral marrow with this probe. At 4 degrees C, the probe bound to the luminal surface of marrow sinus endothelium. The binding was inhibitable in the presence of excess native Fe-TF indicating the specificity of the binding. At 37 degrees C, the probe was internalized largely via a system of coated pits and vesicles and transported across the endothelium via a system of tubules and endosomal vesicles. It could not be ascertained if all Fe-TF was still associated with the colloidal gold probe within the endothelium, but the probe appeared to be externalized on the abluminal side into the interstitium where it subsequently bound to the surface of marrow erythroblasts and was internalized. Endothelium appeared to store part of the probe within a large vesicular system. No transport of Fe-TF was noted through diaphragmed fenestrations, diaphragmed vesicles, or interendothelial junctions. No endothelial uptake of this magnitude was noted when native gold particles or gold-labeled bovine serum albumin was used. Our findings indicate that in the bone marrow, gold-labeled Fe-TF is first taken up by sinus endothelium through a receptor-mediated mechanism and is possibly transported transendothelially via a vesicular system (transcytosis).     

The role of endosomal traffic in the transendothelial transport of ceruloplasmin in the liver

Through a process resembling receptor-mediated endocytosis, liver endothelium binds and internalizes the plasma glycoprotein ceruloplasmin (CP) on the luminal side. The protein is then transported via a vesicular system to the albuminal side where it is externalized to the space of Disse. In its path, the glycoprotein is fully desialylated. To determine if the endosomal compartment is involved in this transport, we used endosomal inhibitors NH4Cl, ethylamine as well as monensin to quantitatively measure the magnitude of radiolabeled CP transport across purified liver endothelial cells. All three reagents inhibited the transport of CP and its discharge by endothelium. The magnitude of inhibition was dose-related for all three reagents. We conclude that the endosomal compartment is involved in the transendothelial transport of CP across the liver endothelium.     

Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate

The role of liver cytosolic fatty acid binding protein (L-FABP) in fatty acid transport and metabolism is unclear. Female liver contains substantially more L-FABP than male liver. Female liver also has a different fatty acid transport phenotype, including more rapid uptake, efflux and cytoplasmic transport. However, it is not known if the greater levels of L-FABP are responsible for these differences. We therefore determined whether increasing L-FABP using clofibrate causes male liver to acquire a female transport phenotype. The multiple indicator dilution (MID) method was used to estimate the rate constants for influx, efflux and cytoplasmic diffusion of palmitate in isolated perfused rat livers. Clofibrate treatment increased cytosolic concentrations of L-FABP 4.2+/-0.8-fold, the rate of cytoplasmic diffusion of palmitate 4.3+/-1.7-fold, and the steady-state palmitate extraction 1.5+/-0.3-fold (mean+/-S.E.). Influx and efflux constants were both increased (by 44% and 79%, respectively) to levels typical of female livers. These data suggest that clofibrate-induced elevation of cytosolic L-FABP not only stimulates intracellular diffusion but also influx and efflux of fatty acids. Possible mechanisms include reducing fatty acid binding to cytoplasmic membranes, induction of membrane fatty acid carriers, and catalyzing fatty acid exchange between aqueous cytoplasm and the plasma membrane.     

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi

The temperature and ATP dependence of transport of the vesicular stomatitis virus strain ts045 G protein from the endoplasmic reticulum (ER) to an early Golgi compartment containing mannosidase I was studied in the mutant Chinese hamster ovary cell clone 15B. Appearance of G protein containing the Man5GlcNAc2 oligosaccharide species occurred after a shift to the permissive temperature with a lag period of 5 min and without detectable formation of the intermediate Man7GlcNAc2 and Man6GlcNAc2 species. Two biochemically distinct transport steps were detected during transport from the ER to the Golgi. An initial step is temperature sensitive, thermoreversible, and requires a high threshold of cellular ATP for maximal rate of transport (80% of the normal cellular ATP pool). Export from the ER is inhibited at 65% of the normal cellular ATP pool. Prolonged incubation at reduced levels of cellular ATP or at the restrictive temperature resulted in the accumulation of G protein in either the Man8GlcNAc2 species or the Man7GlcNAc2 and Man6GlcNAc2 species, respectively. Reversal of the temperature-sensitive block is ATP coupled. A second step is insensitive to incubation at the restrictive temperature and proceeds efficiently when the cellular ATP pool is reduced to 20% of the control. G protein accumulates at this intermediate step during prolonged incubation at 15 degrees C. The data suggest a functional division of processes required for transport of protein between the ER and Golgi compartments. The two steps may reflect the export (budding) and delivery (fusion) of proteins through vesicular trafficking between the ER and Golgi.     

Removal of pleural liquid and protein by lymphatics in awake sheep

The contribution of the parietal pleural lymphatics to pleural liquid and protein removal is unclear. We asked two questions. What is the rate of removal of sterile, artificial hydrothoraxes in awake sheep? What percentage is removed through parietal pleural lymphatics? Three days after the placement of a rib capsule in 18 sheep, we instilled a 10 ml/kg 1.0 g/dl autologous protein solution with labeled albumin and erythrocytes through the capsule into the pleural space. Erythrocytes were used as a marker for lymphatic flow. We measured terminal pleural liquid volume and radioactivity at periods from 2 to 48 h. In three sheep, we obtained a third volume measurement at 6 h by the volume of dilution technique. We found that hydrothorax removal could be described by a linear function with a constant rate: 0.28 +/- 0.01 ml.kg-1.h-1 (mean +/- SE) for the grouped data, and 0.20, 0.28, and 0.31 ml.kg-1.h-1 for the individual sheep. At 24 h, erythrocyte clearance was 89 +/- 16% (mean +/- SD) that of liquid and albumin clearance. We conclude that in awake sheep with large hydrothoraxes, pleural liquid and protein are removed at a rate of 0.28 +/- 0.01 ml.kg-1.h-1 (mean +/- SE) and lymphatics are responsible for at least 89% of this removal.     

Influx and incorporation into protein of L-phenylalanine in the perfused rat pancreas: effects of amino acid deprivation and carbachol.

The rate of protein synthesis in the isolated perfused rat pancreas was measured from the rate of incorporation of L-[3H]phenylalanine into total protein, and was compared with the transport of this amino acid into the epithelium. Unidirectional (15 s) and net (15-30 min) uptake of L-[3H]phenylalanine was measured relative to D-[14C]mannitol (extracellular marker) using a cell loading technique. The fractional rate of protein synthesis in the pancreas was also measured in vivo using a flooding dose technique and found to be 118 +/- 10% day-1 (corresponding to an absolute rate of incorporation of L-Phe into protein of 36.1 +/- 3 nmol min-1 g-1) in overnight fasted rats. Compared with the in vivo rate, the perfused pancreas exhibited a markedly lower rate of protein synthesis which increased significantly when amino acids were added to the perfusate (15.6 +/- 1.9 vs. 22.5 +/- 0.9% day-1 or 4.7 +/- 0.6 vs. 6.9 +/- 0.3 nmol L-Phe min-1 g-1). Carbachol (3 x 10(-7) M) stimulated protein synthesis provided amino acids were also supplied in the perfusate. Protein synthesis rates measured under all conditions in vivo and in vitro were at least an order of magnitude lower than the unidirectional influx (121 +/- 14 nmol min-1 g-1) of L-phenylalanine into the pancreatic epithelium. These results demonstrate that amino acid transport across the basolateral membrane of the epithelium is not rate-limiting for pancreatic protein synthesis.     

Binding and transport of transthyretin-gold by the endothelium of the rat choriocapillaris

The photoreceptors of the neural retina require retinol for synthesis of rhodopsin. In the plasma, retinol is bound to retinol binding protein which is carried by transthyretin (TTR; formerly called prealbumin). It is unknown whether, or how, retinol carrier proteins cross the endothelium of the choriocapillaris, the blood supply to the outer neural retina. This was examined in the present study with TTR-gold probes perfused into rats and localized by electron microscopic techniques. TTR-gold, often in clusters, was localized to diaphragmed fenestrae, parajunctional areas, coated pits, transendothelial channels, multivesicular bodies, and to vesicles close to the Golgi apparatus. The probe was also identified at the luminal and abluminal fronts and the interior of transendothelial channels in an apparent sequence of transit. TTR-gold was also found in a series of interconnected vesicles adjacent to the abluminal side of the endothelium. Localizations were not seen when rat albumin fraction V was substituted for TTR and when the rats were perfused with Pronase E before labeling with TTR-gold. These observations indicate that binding and receptor mediated-like transport of TTR by the endothelium of the choriocapillaris is present. This is similar to the processing of heparin-gold by this endothelium.     

Electron microscopic characteristics of plant peroxidase transport pathways in the microcirculatory bed of the rabbit peritoneum

The dynamics of exogenic peroxidase transfer from blood into the roots of the rabbit mesenteric lymphatic system have been studied by means of electron microscopic methods in combination with the trasser technique. Light optic identification of the vascular segments and selection of samples for electron microscopic analysis make it possible to reveal certain differences in the pathways of protein transport via the walls of the blood capillaries and venules. The vesicular transport is the only means for peroxidase to be transferred via the walls of the mesenteric blood capillaries. The time for transendothelial transfer of the marker is more than 10 min. In the venules the vesicular transport of protein does not differ from that in the capillaries, however, the predominant leakage of peroxidase from blood into the interstitium is performed through open interendothelial contacts. The hemato-interstitial transport via the intercellular clefts takes less than 3 min. For transferring protein from the interstitium into the lumen of the lymphatic capillaries and postcapillaries, the vesicular mechanism is used, and to a less extent--the open intercellular contacts. A suggestion is made that the term "open contact" should be understood in functional meaning and this means should be considered as an intercellular pathway for transporting molecules of a definite size.     

Temperature modulates P2X receptor-mediated cardiovascular responses to muscle afferent activation

Static muscle contraction increases ATP release into the muscle interstitial space. Elevated ATP in muscle stimulates thin fiber muscle afferents and increases blood pressure via engagement of purinergic P2X receptors. In addition, ATP activates P2X receptors and enhances cardiovascular responses induced by stimulation of muscle mechanoreceptors. In this study, we examined whether elevated muscle temperature would attenuate and whether reduced temperature would potentiate P2X effects on reflex muscle responses. alpha,beta-Methylene ATP (alpha,beta-MeATP) was injected into the arterial blood supply of hindlimb muscle to stimulate P2X receptors, and muscle stretch was induced to activate mechanically sensitive muscle afferents as alpha,beta-MeATP was injected in 10 anesthetized cats. Femoral arterial injection of alpha,beta-MeATP (1.0 mM) increased mean arterial pressure (MAP) by 35+/-5 (35 degrees C), 26+/-3 (37 degrees C), and 19+/-3 mmHg (39 degrees C; P<0.05 vs. 35 degrees C), respectively. Muscle stretch (2 kg) elevated MAP. The MAP response was significantly enhanced 34% and 36% when alpha,beta-MeATP (0.2 mM) was arterially infused 5 min before muscle stretch at 35 degrees and 37 degrees C, respectively. However, as muscle temperature reached 39 degrees C, the stretch-evoked response was augmented only 6% by alpha,beta-MeATP injection, and the response was significantly attenuated compared with the response with muscle temperature of 35 degrees and 37 degrees C. In addition, we also examined effects of muscle temperature on alpha,beta-MeATP enhancement of the cardiovascular responses to static muscle contraction while the muscles were freely perfused and the circulation to the muscles was occluded. Because muscle temperature was 37 degrees C, arterial injections of alpha,beta-MeATP significantly augmented contraction-evoked MAP response by 49% (freely perfused) and 53% (ischemic condition), respectively. It is noted that this effect was significantly attenuated at a muscle temperature of 39 degrees C. These data indicate that the effect of P2X receptor on reflex muscle response is sensitive to alternations of muscle temperature and that elevated temperature attenuates the response.