Donald A. Vessey

A spin-label study of the role of phospholipids in the regulation of membrane-bound microsomal enzymes☆

Abstract The activities of two hepatic microsomal enzymes, glucose 6-phosphatase and UDP-glucuronyltransferase, were determined at assay temperatures in the range between 5 °C and 40 °C. Arrhenius plots of the activities of both enzymes display abrupt changes at about 19 °C. An additional discontinuity at 32 °C is observed in the case of UDP-glucuronyltransferase. Only the latter discontinuity is detected in microsomes subjected to partial treatment with phospholipase A. Lipophilic nitroxide radicals were introduced into samples of the same microsomal preparations and the corresponding electron spin resonance spectra were recorded over the same temperature range. Temperature dependence of an empirical spectral parameter, related to the fluidity of the matrix solubilizing the moleoular probes, reveals apparent breaks at 19 °C and 32 °C in intact microsomes. Only the break at 19 °C was observed in microsomes subjected to sonic disruption. No breaks were detected in plots of data measured in microsomes partially treated with phospholipase A. The correlation between the enzymatic data and the data obtained from lipophilic spin-probes is indicative of the dependence of tightly bound membrane enzymes on the physical state of membrane lipids. The relevance of the data to further studies of the protein-lipid interactions is discussed.

Ischaemic postconditioning protects isolated mouse hearts against ischaemia/reperfusion injury via sphingosine kinase isoform-1 activation

AIMS Sphingosine-1-phosphate (S1P) plays a vital role in cytoskeletal rearrangement, development, and apoptosis. Sphingosine kinase-1 (SphK1), the key enzyme catalyzing the formation of S1P, mediates ischaemic preconditioning. Ischaemic postconditioning (POST) has been shown to protect hearts against ischaemia/reperfusion injury (IR). To date, no studies have examined the role of SphK1 in POST. METHODS AND RESULTS Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (+/-dP/dtmax), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and +/-dP/dtmax were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from 40 +/- 2% in the control group to 29 +/- 2% of the risk area in the POST group (P < 0.05, n = 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 +/- 5%) was not different from the KO control group (53 +/- 5% of risk area, n = 4, P = NS). CONCLUSIONS A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1-KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST.

Sphingosine 1-phosphate is an important endogenous cardioprotectant released by ischemic pre- and postconditioning.

Exogenous sphingosine 1-phosphate (S1P) is an effective cardioprotectant against ischemic injury. We have investigated the hypothesis that S1P is also an important endogenous cardioprotectant released during both ischemic preconditioning (IPC) and ischemic postconditioning (IPOST). IPC of ex vivo rat hearts was instituted by two cycles of 3 min ischemia-5 min reperfusion prior to 40 min of index ischemia and then 40 min of reperfusion. IPC resulted in 70% recovery of left ventricular developed pressure (LVDP) upon reperfusion and a small infarct size (10%). VPC23019 (VPC), a specific antagonist of S1P(1 and 3) G protein-coupled receptors (GPCRs), when present during preconditioning blocked protection afforded by two cycles of IPC. VPC also blocked preconditioning of isolated rat cardiac myocytes subjected to hypoxia-reoxygenation injury. Increased release of S1P from myocytes in response to IPC was also demonstrated. These data indicate that S1P is released from myocytes in response to IPC and protects by binding to S1P GPCRs. In the ex vivo heart, if a third cycle of IPC was added to increase release of endogenous mediators, then the need for any individual mediator (e.g., S1P) was diminished and VPC had little effect. The adenosine antagonist 8-(p-sulfophenyl)-theophylline (8-SPT) likewise inhibited protection by two cycles but not three cycles of IPC, but VPC plus 8-SPT inhibited protection by three cycles of IPC. Similar to IPC, IPOST induced by four postindex ischemia cycles of 15 s reperfusion-15 s ischemia resulted in 66% recovery of LVDP and a 7% infarct size. When VPC was present during postconditioning and reperfusion, LVDP only recovered by 26% and the infarct size increased to 27%. Adding an additional cycle of IPOST reduced the inhibitory effect of VPC and 8-SPT individually, but not their combined effect. These studies reveal that S1P is an important mediator of both IPC and IPOST that is released along with adenosine during each cycle of IPC or IPOST.

Regulation of microsomal enzymes by phospholipids: IV. Species differences in the properties of microsomal UDP-glucuronyltransferase

Abstract Modification of the activity of UDP-glucuronyltransferase (UDP-glucuronate glucuronyltransferase (acceptor unspecific), EC 2.4.1.17) by treatment of microsomes with phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) from both Naja naja and Crotalus adamanteus venoms and with p -chloromercuribenzoate has been studied in a variety of species: guinea pig, mouse, rat, beef, rabbit and human. Contrary to reports in the literature that -SH group reagents and phospholipase A inhibit the enzyme, activation of UDP-glucuronyltransferase by these agents was noted in all species examined. Thus, constraint on maximum potential activity is a general property of this enzyme in untreated microsomes from a variety of species. On the other hand, the extent of activation of glucuronyltransferase by treatment of microsomes with phospholipase A or p -chloromercuribenzoate varied with the species studied. In addition to variation in the extent of activation, species differences in the reactivities of the -SH groups associated with UDP-glucuronyltransferase were noted.

Kinetic properties of microsomal UDP-glucuronyltransferase evidence for cooperative kinetics and activation by UDP-N-acetylglucosamine

The kinetics of microsomal UDPglucuronyltransferase (EC 2.4.1.17) with p-nitrophenol and o-aminobenzoate as glucuronyl acceptors deviate from Michaelis-Menten at low concentrations of UDPglucuronic acid in that double reciprocal plots are concave. The deviation from linearity in these plots represents a real property of UPDglucuronyltransferases and does not result from artifacts in the assay due to metabolism of substrates or products in side pathways. Careful analysis of the data indicated that they also cannot be explained by postulating multiple enzymes for the synthesis of each glucuronide. The most reasonable and simplest mechanism which is compatible with all the data is that there is cooperativity in the sequential binding of UDPglucuronic acid to UDPglucuronyltransferase. Thus, the binding of the first molecule of UDPglucuronic acid to the enzyme makes the subsequent binding of UDPglucuronic acid more difficult. Assay of UDPglucuronyltransferase in the presence of UDP-N-acetylglucosamine is associated with a reversible decrease in the apparent K0.5 for UDPglucuronic acid with p-nitrophenol or o-aminobenzoate as aglycone. The binding of UDP-N-acetylglucosamine to UDPglucuronyltransferase also shows anomalous kinetics which appear to be most compatible with negative cooperativity. Despite similarities in their structures there is no overlap in the binding of UDPglucuronic acid and UDP N-acetylglucosamine at the active site and regulatory site of the enzyme.

Combined Sphingosine, S1P and Ischemic Postconditioning Rescue the Heart after Protracted Ischemia

Both sphingosine and sphingosine-1-phosphate (S1P) were able to protect the ex vivo rat heart from ischemia reperfusion injury when added to the perfusion medium at the time of reperfusion after a 40min ischemia (postconditioning). Inhibitor studies revealed distinct mechanisms of protection, with S1P employing a G-protein coupled receptor pathway and sphingosine a cyclic nucleotide dependent protein kinase pathway. However, both restored ischemia-induced depletion of phospho-AKT. Extending the ischemia to 75min reduced protection by both S1P and sphingosine, but protection could be enhanced by employing them in combination. Extending the time of ischemia further to 90min almost eliminated cardioprotection by S1P or sphingosine; and their combination gave only modest protection. However, when S1P plus sphingosine was combined with a novel ramped ischemic postconditioning regimen, left ventricle developed pressure recovered by 66% and there was only a 6% infarct size. The data indicate that detrimental changes are accumulating during protracted ischemia but for up to 90min this damage is not irreversible and hearts can still recover with proper treatment.

Sphingosine can pre- and post-condition heart and utilizes a different mechanism from sphingosine 1-phosphate

Consistent with previous reports, sphingosine at a high concentration (5 μM) was cardiotoxic as evidenced by increased infarct size in response to ischemia/reperfusion in an ex vivo rat heart. Sphingosine 1‐phosphate (S1P) at 5 μM was cardioprotective. However, at a physiologic concentration (0.4 μM) sphingosine as well as S1P was effective in protecting the heart from ischemia/reperfusion injury both when perfused prior to 40 min of ischemia (preconditioning) or when added to reperfusion media following ischemia (postconditioning). Protection by sphingosine and S1P was evidenced with both pre‐ and post‐conditioning by a >75% recovery of left ventricular developed pressure during reperfusion and a decrease in infarct size from 45% of the risk area to less than 8%. When VPC23019, an S1P1and3G‐protein coupled receptor antagonist, was added to the preconditioning or postconditioning medium along with S1P, it completely blocked S1P‐induced protection. However, VPC 23019 did not affect the ability of 0.4 μM sphingosine to either precondition or postcondition hearts. Studies of preconditioning revealed that inhibition of protein kinase C with GF109203X blocked preconditioning by S1P. However, GF109203X did not affect preconditioning by 0.4 μM sphingosine. Likewise, cotreatment with the PI3 kinase inhibitor wortmanin blocked preconditioning by S1P but not by sphingosine. By contrast, inhibition of protein kinase G with KT5823 had no effect on S1P preconditioning but completely eliminated preconditioning by sphingosine. Also, the protein kinase A inhibitory peptide 14–22 amide blocked preconditioning by sphingosine but not S1P. These data reveal for the first time that sphingosine is not toxic at physiologic concentrations but rather is a potent cardioprotectant that utilizes a completely different mechanism than S1P; one that is independent of G‐protein coupled receptors and utilizes cyclic nucleotide‐dependent pathways.

Regulation of microsomal enzymes by phospholipids VI. Abnormal enzyme-lipid interactions in liver microsomes from gunn rats

Abstract The rates of synthesis of some glucuronides by liver microsomes from the Gunn strain of rat are abnormally low, but previous investigators of the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase (UDPglucuronate glucuronyltransferase, EC 2.4.1.17) have reported normal levels of activity in these animals. Data presented in this paper indicate, however, that this enzyme is abnormal in Gunn rats. Thus, treatment of liver microsomes from normal Wistar rats with phospholipase A (EC 3.1.1.4) or Triton X-100 increases the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase 10- and 20-fold, respectively, but these agents do not alter activity in microsomes from homozygous Gunn rats. Similarly, phospholipase A and Triton X-100 activate the o-aminophenol and o-aminobenzoate metabolizing forms of UDPglucuronyltransferase in microsomes from normal rats, but are without effect on the enzyme in microsomes from Gunn rats. In contrast, the rates of synthesis of o-aminophenyl- and o-aminobenzoylglucuronides are increased several fold by addition of diethylnitrosamine to microsomes from Gunn rats indicating that the maximum potential activities of UDPglucuronyltransferases are constrained in liver microsomes from both normal and Gunn rats. These data indicate that assays of UDPglucuronyltransferase in native microsomes are not sufficient for delineating the full extent of the defect in the Gunn rat, that there are defects in the function of at least two proteins in liver microsomes from these animals, and that there are abnormal interrelations between some forms of microsomal UDPglucuronyltransferase and their phospholipid environments.

A Sphingosine Kinase Form 2 Knockout Sensitizes Mouse Myocardium to Ischemia/Reoxygenation Injury and Diminishes Responsiveness to Ischemic Preconditioning

Sphingosine kinase (SphK) exhibits two isoforms, SphK1 and SphK2. Both forms catalyze the synthesis of sphingosine 1-phosphate (S1P), a sphingolipid involved in ischemic preconditioning (IPC). Since the ratio of SphK1 : SphK2 changes dramatically with aging, it is important to assess the role of SphK2 in IR injury and IPC. Langendorff mouse hearts were subjected to IR (30 min equilibration, 50 min global ischemia, and 40 min reperfusion). IPC consisted of 2 min of ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), ± dP/dtmax, and heart rate between SphK2 null (KO) and wild-type (WT) hearts. In KO hearts, SphK2 activity was undetectable, and SphK1 activity was unchanged compared to WT. Total SphK activity was reduced by 53%. SphK2 KO hearts subjected to IR exhibited significantly more cardiac damage (37 ± 1% infarct size) compared with WT (28 ± 1% infarct size); postischemic recovery of LVDP was lower in KO hearts. IPC exerted cardioprotection in WT hearts. The protective effect of IPC against IR was diminished in KO hearts which had much higher infarction sizes (35 ± 2%) compared to the IPC/IR group in control hearts (12 ± 1%). Western analysis revealed that KO hearts had substantial levels of phosphorylated p38 which could predispose the heart to IR injury. Thus, deletion of the SphK2 gene sensitizes the myocardium to IR injury and diminishes the protective effect of IPC.

Pannexin-I/P2X 7 Purinergic Receptor Channels Mediate the Release of Cardioprotectants Induced by Ischemic Pre- and Postconditioning

Ischemic pre- and postconditioning protect ex vivo rat hearts from ischemia/reperfusion injury by promoting the release of cardioprotective agents by an unknown mechanism. Because P2X 7 purinergic receptors are known to combine with pannexin-1 to form channels that allow adenosine triphosphate (ATP) release from cells, we hypothesized that these channels have a role in the release of multiple cardioprotectants during ischemic preconditioning (IPC). Addition of either a pannexin-1 hemichannel blocker (5 μmol/L carbenoxolone [CBX] or 0.4 μmol/L mefloquine [MF]) or a selective antagonist of the rat P2X7 purinergic receptor (2 μmol/L brilliant blue G [BBG]) blocked IPC. These antagonists also blocked ischemic postconditioning. Preconditioning by exogenous addition of either sphingosine-1-phosphate or adenosine was not blocked by either CBX or BBG, indicating that they only affected the release of endogenous mediators, not any subsequent steps. To determine if only ATP release was mediated by pannexin-1/P2X7 channels, we added an extra cycle of IPC to release sufficient quantities of additional cardioprotectants to eliminate the dependence on adenosine derivatives. This did not override the inhibition of IPC by CBX or MF, suggesting that the channel mediates the release of multiple cardioprotectants. Inhibitors of other P2X receptors, P2Y receptors, or connexins did not affect IPC. We conclude that a pannexin-1/P2X7 channel is responsible for the release of cardioprotectants induced by ischemic pre- and postconditioning.