Glyburide-reversible cardioprotective effects of calcium-independent phospholipase A2 inhibition in ischemic rat hearts.

OBJECTIVES: A myocardial calcium-independent PLA2 has been described that is activated during myocardial ischemia and this enzyme may modulate ATP-sensitive potassium channels (KATP). The aim of this study was to determine the effect of an inhibitor of this enzyme, a bromoenol lactone, in isolated globally ischemic rat hearts. METHODS: Isolated rat hearts were treated for 10 min with 0.3-6 microM bromoenol lactone and then subjected to 25 min ischemia and 30 min reperfusion. RESULTS: The bromoenol lactone significantly increased coronary flow in nonischemic myocardium, and slightly reduced cardiac function at 6 microM. During global ischemia, time to contracture was significantly increased from vehicle group values in the presence of the bromoenol lactone (EC50 = 1.2 microM). During reperfusion, a concentration-dependent increase in function and a reduction in LDH release were observed for the PLA2 inhibitor. The concentrations of the PLA2 inhibitor which were significantly cardioprotective, inhibited this enzyme in membrane fractions of rat myocardium (IC50 = 0.87 microM). The KATP blocker sodium 5-hydroxydecanoate (5-HD) inhibited the increase in time to contracture observed for the bromoenol lactone. During reperfusion, 5-HD abolished the protective effects of the bromoenol lactone on cardiac function and LDH release. Glyburide had similar effects on the cardioprotective activity of the bromoenol lactone, although it only partially abolished the LDH reducing effect of this agent. CONCLUSIONS: The bromoenol lactone protects ischemic myocardium at concentrations which also inhibit calcium-independent PLA2. This cardioprotection can be attenuated by blockers of KATP, suggesting a potential mechanism for modulation of myocardial KATP.

ATP-sensitive potassium channels and skeletal muscle function in vitro.

Compounds that act at ATP-modulated potassium channels (KATP) were tested in an in vitro model of skeletal muscle ischemia. The extensor digitorum longus muscles were removed from anesthetized rats and placed in tissue baths, and contractions were elicited by electrical field stimulation at 0.2 Hz. During normoxia, the force of contraction gradually decayed to about 55% of the peak over 85 min. None of the KATP openers tested, cromakalim (300 microM), P-1075 (10 microM) and pinacidil (100 microM), affected twitch force during normoxia. However, when the muscles were made anoxic, all three compounds greatly accelerated the loss of function in a concentration-related manner. For example, the cromakalim/vehicle ratios of the area under the force-time curve during anoxia were 0.98 +/- 0.03, 0.77* +/- 0.03 and 0.72* +/- 0.04 for cromakalim at 30, 100 and 300 microM, respectively (*P < .05). Upon reoxygenation, muscles treated with the KATP openers recovered twitch force to a greater extent than those treated with vehicle. Glyburide (1 or 10 microM) had no effect on its own, but it was able to prevent fully the effects of KATP openers during both anoxia and reoxygenation, indicating that the effects of the KATP openers were mediated by KATP. These results suggest that KATP openers would not be beneficial in the treatment of skeletal muscle ischemia in vivo but that they may be useful in preserving skeletal muscle function in cases of ischemia followed by reperfusion.

Activation of a G protein in mouse sperm by the zona pellucida, an egg-associated extracellular matrix.

Mammalian sperm possess a guanine nucleotide-binding regulatory protein (G protein), with properties similar to Gi, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)-mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates Gi proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high-affinity GTPase activity and GTP gamma [35S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm-associated Gi protein. An increase in GTP hydrolysis (approximately 50% over basal activity) and GTP gamma [35S] binding (approximately 25-60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [32P]ADP-ribosylation of a Mr = 41,000 sperm Gi protein, suggesting that the increase in GTPase activity and GTP gamma [35S] binding is associated with the activation of a PT-sensitive sperm G protein(s). The ability of the ZP to stimulate high-affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface-associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.

Signal transduction processes leading to acrosomal exocytosis in mammalian spermatozoa.

Many components of intercellular signaling involved in species-specific sperm binding to the egg's extracellular matrix, the zona pellucida, and the induction of acrosomal exocytosis, an absolute prerequisite to successful fertilization, have properties similar to intercellular signaling mechanisms controlling somatic cell function. Sperm-associated receptors for zona pellucida glycoproteins have been postulated to serve as the initial components of signal transduction cascades leading to the stimulation of cellular effector systems that modulate sperm function. Such receptor-effector systems appear to be coupled through guanine nucleotide-binding regulatory proteins (G proteins).

Chemoattractant-stimulated GTPase activity is decreased on membranes from polymorphonuclear leukocytes incubated in chemoattractant.

Polymorphonuclear leukocytes, PMNs, incubated in a chemoattractant undergo a time-dependent decrease in responsiveness to the chemoattractant; i.e. they desensitize or adapt. We have examined the role of ligand-induced changes at early steps in signal transduction for adaptation of PMNs to chemoattractants. The chemoattractant stimulation of a pertussis toxin-sensitive GTPase activity on PMN membranes was used as an assay of signal transduction. We find a decreased basal GTPase activity and a decrease in the ability of N-formylnorleucylleucylphenylalanine (FN-LLP) to stimulate this activity on membranes prepared from PMNs incubated with the chemotactic peptide FNLLP. The basal GTPase activity is decreased by up to 70% and the peptide-stimulated GTPase activity by up to 95% on membranes from PMNs incubated for 20 min at 37 degrees C in 10(-7) M FNLLP. The decrease in peptide-stimulated GTPase activity cannot be accounted for by the decreased number of FNLLP receptors on the membranes. Rather, receptors that remain available for binding stimulate the GTPase activity with a decreased efficiency. The ligand-induced change in GTPase activity is not stimulus specific. GTPase activity stimulated by both C5a and LTB4 was decreased on membranes from PMNs incubated in FNLLP. The decrease in chemoattractant-stimulated GTPase activity is partially reversed if cells are subsequently incubated at 37 degrees C in the absence of peptide prior to membrane preparation. We detected no quantitative or qualitative change in either pertussis toxin substrates or immunoreactive G proteins when membranes from control and FNLLP-treated cells were compared.

Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels.

Chemotactic factors stimulate the rate of locomotion of polymorphonuclear leukocytes (PMNs). To investigate the importance of cytoplasmic calcium we have examined the ability of the chemotactic peptide N-formylnorleucyl eucylphenalanine (FNLLP) to stimulate the locomotion of PMNs whose cytoplasmic calcium levels were reduced by incubation in EGTA or in EGTA plus the calcium ionophores, ionomycin or A23187. Locomotion was assayed by migration through micropore filters and by time-lapse videomicroscopy. Cells in EGTA exhibited similar or slightly reduced rates of locomotion compared to cells in Hanks' balanced salt solution (HBSS). The peptide dose dependence for the stimulation of locomotion was similar in medium containing calcium or EGTA. The presence of 1 microM ionophore plus EGTA had no effect on the stimulation of locomotion by peptide. The presence of ionophores (1 microM) plus external calcium inhibited locomotion.

Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide.

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.

Regulation of mouse oocyte maturation: involvement of cyclic AMP phosphodiesterase and calmodulin.

A decrease in mouse oocyte cAMP occurs during commitment to resume meiosis (R. M. Schultz, R. R. Montgomery, and J. R. Belanoff, 1983, Dev. Biol. 97, 264-273). Experiments described in this report were performed to ascertain if oocyte cyclic nucleotide phosphodiesterase (PDE) is involved in this decrease. PDE activity was found in extracts of mouse oocytes. The activity appeared soluble and not membrane bound. For each of three different PDE inhibitors, a positive correlation was found between the ability of increasing concentrations of each compound to inhibit PDE in oocyte extracts and to inhibit germinal vesicle breakdown (GVBD). Moreover, the more potent the PDE inhibitor, the more effectively it inhibited GVBD. The possibility that calmodulin (CaM) plays a role in maturation was examined since CaM modulates PDE activity in other systems. About 0.3% of total oocyte protein is CaM as determined by radioimmunoassay and activation of exogenous PDE. A CaM-dependent step in maturation was suggested since the CaM inhibitors trifluoperazine and calmidizolium inhibited GVBD in a dose-dependent manner. In addition, the CaM inhibitors W7 and W13 inhibited GVBD at lower concentrations than the less-active corresponding congeners W5 and W12. Oocyte extracts contained a CaM-modulated PDE. Activity was inhibited about 50% by addition of EGTA, and fully restored by addition of exogenous CaM and excess calcium. cAMP hydrolysis was inhibited in a dose-dependent manner by either trifluoperazine, calmidizolium, or W7; maximal inhibition was also about 50%. CaM-modulated PDE, however, did not appear to be the target for the effects of CaM inhibitors on GVBD, since concentrations of W7 that inhibited maturation did not inhibit cAMP hydrolysis in the oocyte. Results from these studies suggest that oocyte PDE is involved in the decrease in cAMP associated with resumption of meiosis, but that the CaM-dependent step occurs subsequent to or concurrently with the drop in cAMP.