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There is now strong evidence that an endothelial mechanism, other than nitric oxide or prostacyclin, exists for dilating arteries and arterioles. This third pathway has been named endothelium-derived hyperpolarizing factor (EDHF) and should not be confused with endothelium-derived relaxing factor, which is nitric oxide. Currently, there are several ideas for the mechanism of EDHF, which may vary among vessels of different organs and species. During some pathologic states, EDHF can be up-regulated. This up-regulation often occurs as the dilator effects of endothelium-derived nitric oxide are suppressed. The up-regulated EDHF may serve in a protective capacity to help maintain blood flow to organs and tissues during these stressful states. Many anesthetics attenuate the dilator actions of EDHF; however, the full clinical implications of this anesthetic-related attenuation are not known. Like its cousins, nitric oxide and prostacyclin, EDHF is an important regulator of blood flow and should prove to be an important clinical consideration as we gain more knowledge of its mechanisms of action.

Elevations in arterial pressure associated with hypertension, microgravity, and prolonged bed rest alter cerebrovascular autoregulation in humans. Using head-down tail suspension (HDT) in rats to induce cephalic fluid shifts and elevate arterial pressure, this study tested the hypothesis that 2-wk HDT enhances cerebral artery vasoconstriction and that an enhanced vasoconstriction described in vitro will alter regional cerebral blood flow (CBF) and vascular resistance (CVR) during standing and head-up tilt. To test this hypothesis, basal tone and vasoconstrictor responses to increases in transmural pressure, shear stress, and K+ were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 10−5 M) and with endothelium removal. Endothelial NOS (eNOS) mRNA and protein expression levels were measured by RT-PCR and immunoblot, respectively. Regional CBF and CVR were determined with a radiolabeled tracer technique and quantitative autoradiography. Basal tone and all vasoconstrictor responses were greater in MCAs from HDT rats. l-NAME and endothelium removal abolished these differences between groups, and HDT was associated with lower levels of MCA eNOS protein. CBF in select regions was lower and CVR higher during standing and head-up tilt in HDT rats. These results indicate that chronic cephalic fluid shifts enhanced basal tone and vasoconstriction through alterations in the eNOS signaling mechanism. The functional consequence of these vascular alterations with HDT is regional elevations in CVR and corresponding reductions in cerebral perfusion.

Background and Purpose— The contribution of endothelium-derived hyperpolarizing factor (EDHF) to ATP-mediated dilations is significantly attenuated in the rat middle cerebral artery of intact and estrogen-treated ovariectomized (OVX) females compared with males and vehicle-treated OVX females. Since an increase in endothelial calcium appears to be a critical prerequisite in the EDHF response, we tested the hypothesis that endothelial cell intracellular calcium ([Ca 2+ ] i ) fails to reach sufficient levels to elicit robust EDHF-mediated dilations in females and that this effect is mediated by estrogen. Methods— Vascular diameter and [Ca 2+ ] i were measured concomitantly in perfused middle cerebral artery segments with the use of videomicroscopy and fura 2 fluorescence, respectively. Results— In the presence of N G -nitro- l -arginine methyl ester and indomethacin, the dilation to 10 −5 mol/L ATP was significantly reduced ( P 2+ ] i increased to comparable levels in intact females (461±116 nmol/L), estrogen-treated OVX females (417±50 nmol/L), intact males (421±77 nmol/L), and vehicle-treated OVX females (530±92 nmol/L). In response to luminal ATP (10 −5 mol/L), smooth muscle cell [Ca 2+ ] i decreased to a greater degree in males (37±4%; n=8) compared with females (21±5%; n=7) and in vehicle-treated OVX females (18±7%; n=7) compared with estrogen-treated OVX females (3±5%; n=9). Conclusions— Our data suggest that loss of a factor coupling EDHF to reduction of ionized smooth muscle cell [Ca 2+ ] i accounts for the attenuated EDHF-mediated dilations in the female middle cerebral artery.

In cerebral vessels, there is at least one factor or mechanism not involving nitric oxide/endothelium-derived relaxing factor (NO/EDRF) or prostacyclin that is responsible for dilations when endothelial receptors are stimulated. This factor or mechanism is termed endothelium-derived hyperpolarizing factor (EDHF). In rat middle cerebral arteries, branches of the middle cerebral arteries and penetrating arterioles, ATP or UTP elicits the release of EDHF through stimulation of P2Y 2 receptors on the endothelium. The dilations are characterized by the involvement of calcium-activated potassium channels (K Ca ) and hyperpolarizations in vascular smooth muscle. In smaller arteries and in arterioles, EDHF appears to be more important than in larger cerebral arteries. Finally, the EDHF response is upregulated 24 h following mild traumatic brain injury (TBI). EDHF may play an important role in the regulation of cerebral blood flow during normal physiological conditions and an even greater role following pathological conditions such as TBI.

Evidence in the literature suggests that endothelium-derived hyperpolarizing factor (EDHF) may act in a compensatory manner such that during conditions of compromised nitric oxide (NO), EDHF serves as a back-up mechanism. Given that constitutive NO synthase is chronically downregulated after head trauma, we tested the hypothesis that EDHF is potentiated following injury. Male adult rats were subjected to either sham injury (n = 27) or mild controlled cortical impact (CCI) injury (n = 26). Branches of the middle cerebral artery (MCA) directly within the contusion site were harvested either 1 or 24 h later, pressurized to 60 mm Hg in a vessel chamber and allowed to develop spontaneous tone. Relaxation to luminal application of adenosine triphosphate (ATP) was similar in all groups. Relaxation to ATP in the presence of L-NAME (N(G)-nitro-L-arginine methyl ester) and indomethacin was similar in all groups except for vessels isolated at 24 h following mild CCI injury. In this case, L-NAME and indomethacin had no effect on the ATP-mediated dilation. The ATP-mediated dilation in L-NAME and indomethacin-treated MCA branches was inhibited by charybdotoxin, an inhibitor of large conductance Ca2+-sensitive K+ channels. These findings suggest that there is a significant potentiation of the EDHF-mediated dilation to ATP in cerebral arteries isolated at 24 h following mild CCI injury.

N-methyl-D-aspartate (NMDA) receptor antagonists have been demonstrated widely to be neuroprotective in cerebral ischemia, hypoxia, and traumatic brain injury. However, although noncompetitive NMDA antagonists have typically proven efficacious under all of these conditions, competitive antagonists have not been shown to be beneficial following moderate traumatic brain injury. The present study has used phosphorus magnetic resonance spectroscopy ([31P]MRS) to examine the effects of the competitive antagonist cis-4-(phosphonomethyl) piperidine-2-carboxylic acid (CGS-19755) and the noncompetitive antagonist dextromethorphan on biochemical outcome following fluid percussion-induced traumatic brain injury in rats. Five minutes prior to induction of moderate (2.8 +/- 0.2 atm) fluid percussion brain injury, animals received either CGS-19755 (10 mg/kg iv), dextromethorphan (10 mg/kg iv), or equal volume saline vehicle. [31P]MRS spectra were then acquired for 4 h post-trauma and intracellular pH, free magnesium concentration, cytosolic phosphorylation potential, and oxidative capacity determined. Both CGS-19755-treated animals and saline treated controls demonstrated significant and sustained declines in intracellular free magnesium concentration and bioenergetic status following trauma. In contrast, administration of dextromethorphan significantly attenuated free magnesium decline and improved bioenergetic state during the post-traumatic monitoring period. These results suggest that the neuroprotective actions of NMDA antagonists following traumatic brain injury are associated with attenuation of free magnesium decline and that such actions seem to be preferentially mediated by noncompetitive blockers.

It is well established that stimulation of receptors on the endothelium can elicit dilation of arteries and arterioles by initiating the synthesis and release of nitric oxide (NO) and/or metabolites of the cyclooxygenase pathway (most often prostacyclin). Recent evidence now suggests that there is at least one other endothelium-dependent dilator mechanism that does not involve NO or a cyclooxygenase metabolite. This mechanism has been termed “endothelium-derived hyperpolarizing factor” (EDHF). (Note that EDHF is different from endothelium-derived relaxing factor [EDRF], which is often associated with NO. It should also be noted that recent studies suggest that EDHF may not be a “factor” but rather a process or mechanism. To be more accurate, the term “EDHF” should be referred to as a non-NO, noncyclooxygenase endothelium-dependent hyperpolarization. However, in order to maintain consistency in the literature, we will refer to it as “EDHF.”) We believe that EDHF is a major regulator of cerebral blood flow during physiological states and may become even more important following pathological insults such as ischemia or traumatic brain injury. The purpose of this editorial is to familiarize the reader with EDHF and to highlight the potential importance of EDHF in the cerebral circulation. While the defining criteria for EDHF or the mechanism of endothelium-dependent hyperpolarizations can vary, we will characterize the process as (1) requiring endothelium, (2) being distinct from NO and a cyclooxygenase metabolite, (3) hyperpolarizing the vascular smooth muscle (VSM), and (4) involving calcium-activated potassium (KCa) channels. The existence of a third pathway was alluded to in the late 1980s, when some endothelium-dependent relaxations were shown to be resistant to inhibitors of NO and cyclooxygenase.1,2⇓ Although it has been suggested that EDHF-dependent dilations reflect incomplete inhibition of nitric oxide synthase (NOS),3 there is now convincing evidence of the existence of a non-NO, …

We tested the hypotheses that EDHF in rat middle cerebral arteries (MCAs) involves 1) metabolism of arachidonic acid through the epoxygenase pathway, 2) metabolism of arachidonic acid through the lipoxygenase pathway, or 3) reactive oxygen species. EDHF-mediated dilations were elicited in isolated and pressurized rat MCAs by activation of endothelial P2Y2receptors with either UTP or ATP. All studies were conducted after the inhibition of nitric oxide synthase and cyclooxygenase with Nω-nitro-l-arginine methyl ester (10 μM) and indomethacin (10 μM), respectively. The inhibition of epoxygenase with miconazole (30 μM) did not alter EDHF dilations to UTP, whereas the structurally different epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanoic acid (20 or 40 μM) only modestly inhibited EDHF at the highest concentration of UTP. An antagonist of epoxyeicosatrienoic acids, 14,15-epoxyeicosa-5( Z)-enoic acid, had no effect on EDHF dilations to UTP. Chronic inhibition of epoxygenase in the rat with 1-aminobenzotriazol (50 mg/kg twice daily for 5 days) did not alter EDHF dilations. The inhibition of the lipoxygenase pathway with either 10 μM baicalein or 10 μM nordihydroguaiaretic acid produced no major inhibitory effects on EDHF dilations. The combination of superoxide dismutase (200 U/ml) and catalase (140 U/ml) had no effect on EDHF dilations. Neither tiron (10 mM), a cell-permeable scavenger of reactive oxygen species, nor deferoxamine (1 or 10 mM), an iron chelator that blocks the formation of hydroxyl radicals, altered EDHF dilations in rat MCAs. We conclude that EDHF dilations in the rat MCA do not involve the epoxygenase pathway, lipoxygenase pathway, or reactive oxygen species including H2O2.

In the middle cerebral artery (MCA), the presence of nitric oxide (NO) is responsible for maintaining a more dilated state than in its absence during increases in extracellular K(+) and osmolality. The purpose of the present study was to determine whether the involvement of NO was due to (a) a direct effect of the K(+)/osmolality (K(hyper)) on the endothelium or (b) a 'permissive' role of NO. MCAs (approximately 210 microm o.d.) were isolated, cannulated with glass micropipettes, and pressurized to 85 mmHg. When K(+) (KCl) in the extraluminal bath was increased to 21 mM, the diameter increased by 15-20% with the magnitude of dilation diminishing with further increases in K(hyper). The addition of N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-5) mM), an inhibitor of nitric oxide synthase, had no significant effect on dilations at lower K(hyper) concentrations but constricted the arteries relative to the control at 51, 66, and 81 mM K(hyper). In the presence of L-NAME, the addition of an exogenous NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-8) M) or an analog of cGMP, 8-bromo-cGMP (6x10(-5) M), tended to restore the response of K(hyper)to near the original response. We conclude that the basal release of NO from the endothelium plays a permissive role in the K(hyper)-induced response.

After traumatic brain injury, extracellular K(+) in brain can dramatically increase. We studied the effects of increased K(+) on the isolated pressurized rat middle cerebral artery (MCA). MCAs (200-250 microm OD) were isolated, cannulated with glass micropipettes, and pressurized. K(+) was increased in the extraluminal bath using three paradigms: (1) isotonic K(+) (K(iso)) where increases in K(+) were offset by decreases in Na(+), (2) hypertonic K(+) (K(hyper)) where K(+) was increased without a concomitant adjustment of Na(+), and (3) K(suc), a solution using K(iso) but with the addition of sucrose to obtain a hypertonic solution. Increases in K(+) in the extraluminal bath produced significant dilations (approximately 20%) at 21 mM K(+) in all three groups (K(iso), K(hyper), and K(suc)). With the K(hyper) and K(suc) groups, the magnitude of the dilation diminished with further increases in K(+). L-NAME (10(-5) M), an inhibitor of nitric oxide synthase, had no effect on the response of the K(hyper) and K(suc) groups at 21 mM but significantly enhanced constrictions of the MCAs above 40 mM K(+) compared to the control. The K(iso) group was not affected by L-NAME at any K(+) concentration and showed profound constrictions above 40 mM K(+). We conclude that changes in the K(+) concentration and osmolality of the extracellular fluid may have profound effects on the cerebral vasculature.

Using an open cranial window technique, the authors investigated the mechanisms associated with the suppressed CO2 reactivity after mild controlled cortical impact (CCI) injury in rats. The dilation of arterioles (n = 7) to hypercapnia before injury was 38 ± 12%, which was significantly reduced both at 1 hour (23 ± 15% dilation) and at 2 hours after injury (11 ± 19% dilation). In the presence of L-arginine (10 mmol/L topical suffusion, 300 mg/kg intravenous infusion), the dilation of pial arterioles (n = 6) to hypercapnia was partially restored to 30 ± 6% at 2 hours after injury. In the presence of the nitric oxide (NO) donor, S-nitroso- N-acetylpenicillamine (SNAP) (10−8 mol/L topical suffusion), the dilation of pial arterioles (n = 5) to hypercapnia remained diminished (5 ± 7%) at 2 hours after injury. The dilation of pial arterioles (n = 4) to hypercapnia also remained suppressed (5 ± 6%) with topical suffusion of the free radical scavengers, polyethylene glycol-superoxide dismutase (60 units/mL) and polyethylene glycol-catalase (40 units/mL). The authors have shown that L-arginine at least partially restores the diminished response to hypercapnia after mild CCI injury. Furthermore, these data suggest that the beneficial effects of L-arginine are mediated by a combination of providing substrate for NO synthase and scavenging free radicals.

Comparative physiologists and biochemists working with tissues at varying temperatures and ionic strength are required to adjust apparent equilibrium constants (K′) of biochemical reactions to the experimental conditions prior to calculating cytosolic bioenergetic parameters (transformed Gibbs free energy of formation, fΔG′ATP; cytosolic phosphorylation ratio, [ATP]/[ADP][Pi]; [phosphocreatine]:[orthophosphate] ratio [PCr]/[Pi]) and kinetic parameters (free [ADP], [Pi] and [AMP]). The present study shows how to adjust both K′ and the equilibrium constants of reference reactions (Kref) of creatine kinase (ATP: creatine N-phosphotransferase; EC 2.7.3.2), adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) and adenosinetriphosphatase (ATP phosphohydrolase; EC 3.6.1.3) to temperature and ionic strength. This information, together with our previous study showing how to adjust equilibria to varying pH and pMg, is vital for the quantification of organ and tissue bioenergetics of ectotherms and endotherms under physiological conditions.

Phosphorus magnetic resonance spectroscopy ( 31 P MRS) is a noninvasive technique that has been used to estimate free intracellular magnesium concentration (free [Mg 2+ ]). Free [Mg 2+ ] is computed from the chemical shift separation between the α- and β-phosphate resonances of ATP. The current study was undertaken to critically assess the influence of noise effects in estimating free [Mg 2+ ] in rat brain subjected to moderate parasagittal fluid percussion-induced injury. We show that contrary to published data, free [Mg 2+ ] does not significantly change for up to 4 h after moderate trauma in different rat strains and using different surface coils. Before injury, free [Mg 2+ ] = 0.56 ± 0.11 (mean ± SD, n = 36) and 4 h post-trauma, free [Mg 2+ ] = 0.56 ± 0.28. Out results suggest that explanations for this discrepancy comprise errors of chemical shift assignments accompanying low signal-to-noise ratios and the method of analysis employed. Indeed, the authors propose that spectra of β-ATP signal-to-noise ratio less than 5 :1 will produce significant noise-induced errors. We conclude that without knowledge of the inherent errors in 31 P MRS spectroscopy and appropriate statistical analysis, caution should be exercised in calculating free [Mg 2+ ] and using these changes as a basis for proposing pharmacotherapeutic interventions.

Physiologists and biochemists frequently ignore the importance of adjusting equilibrium constants to the ionic conditions of the cell prior to calculating a number of bioenergetic and kinetic parameters. The present study examines the effect of pH and free magnesium levels (free [Mg2+]) on the apparent equilibrium constants (K′) of creatine kinase (ATP: creatine N-phosphotransferase; EC 2.7.3.2), adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) and adenosinetriphosphatase (ATP phosphohydrolase; EC 3.6.1.3) reactions. We show how K′ can be calculated using the equilibrium constant of a specified chemical reaction (Kref) and the appropriate acid-dissociation and Mg2+-binding constants at an ionic strength (I) of 0.25 mol l−1and 38 °C. Substituting the experimentally determined intracellular pH and free [Mg2+] into the equation containing a known Kref and two variables, pH and free [Mg2+], enables K′ to be calculated at the experimental ionic conditions. Knowledge of K′ permits calculation of cytosolic phosphorylation ratio ([ATP]/[ADP][Pi]), cytosolic free [ADP], free [AMP], standard transformed Gibbs energy of formation (ΔfG′ °ATP) and the transformed Gibbs energy of the system (ΔfG′ATP) for the biological system. Such information is vital for the quantification of organ and tissue bioenergetics under physiological and pathophysiological conditions.

The current paper develops a simple mathematical model for the determination of the concentration of ionic species using the chemical shifts of ATP from phosphorus magnetic resonance spectroscopy (31P MRS) spectra. An iterative method is used to evaluate the equilibrium constants for a range of Mg/ATP equilibria. Initially, an expression for free magnesium concentration is determined by consideration of four equilibria. We then demonstrate how any number of equilibria may be included using this simplified analysis, by incorporation of pseudo equilibrium constants. This approach may be applied to the interpretation of both ex vivo and, more importantly, in vivo MRS spectra. Furthermore, the authors suggest that the commonly used equation for determining free intracellular magnesium concentration as presented by Gupta and colleagues is an oversimplification, particularly under conditions of fluctuating pH.