Protective influence of phosphocreatine (Neoton) on the neural structures of the heart and brain

In acute experiments on dogs, we demonstrated that local immunogenic injury to the heart resulting from injection of anticardial cytotoxic serum is accompanied by suppression of a vagus-mediated depressor reflex evoked by intracoronary injection of 5 μg veratrine. Preliminary i.v. injection of 250 mg/kg phosphocreatine to a significant extent prevented the development of immunogenic heart injury and served to normalize the cardiogenic depressor reflex (we measured the heart rate, systemic arterial pressure, pressure in the left ventricle, and its first derivative, and also recorded the afferent activity in the cardial branches of the vagus nerve). These data are indicative of a protective effect of phosphocreatine on the receptor and afferent structures in the heart. At the same time, a parallel study of the effects of application of phosphocreatine on the spike activity of single neurons and on evoked potentials in the neocortex of rats showed that phosphocreatine increases the excitability of cortical neurons by facilitating the processes of synaptic transmission. This was manifested in an increase in the frequency of background spike activity of the neurons and in facilitation of the development of epileptiform reactions evoked by surface application of penicillin after preliminary applications of phosphocreatine.     

Cardiogenic reflexes after immune injury of the heart

Afferent and efferent spike activity from the parasympathetic (vagus) and sympathetic cardiac nerves were recorded simultaneously with ECG, and indices of heart function were measured in acute experiments on anesthetized dogs, which allowed us to study the modifications of cardio-cardiac reflex influences after a local immune heart injury. After an injury nidus has been formed in the heart, cardiogenic depressor reflexes evoked by an intracoronary application of veratrine or bradykinin were considerably suppressed or even abolished, and afferent spike activity in the vagus cardiac nerves noticeably decreased. At the same time, both the facilitation of activity in sympathetic afferent fibers and pressor reflex effects were preserved after the heart injury. Different localization of vagus and sympathetic afferent structures in the heart and their specialized sensitivity to the biologically active substances are suggested as the factors determining the pattern of cardiogenic reflex influences after a heart injury.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 18–25, January–February, 1995.     

Effect of indomethacin-induced inhibition of arachidonic acid metabolism on the development of cardiogenic reflexes

In acute experiments on anesthetized cats, afferent spike activity from the parasympathetic (vagal) and sympathetic cardiac nerves, ECG, and cardiodynamic indices were recorded. The effects of indomethacin-induced blockade of cyclo-oxygenase pathway in metabolism of arachidonic acid on the development of cardiogenic reflex responses after intracoronary injections of veratrine, bradykinin, or prostacyclin were tested. It was found that after indomethacin injection depressor cardiogenic vagal reflexes, evoked by veratrine or bradykinin administrations, became significantly suppressed or practically disappeared. This was accompanied by a drop in the frequency of afferent vagal activity in the cardiac nerves. This effect could be observed throughout the entire period of influence of indomethacin (about 2 h after its injection). Veratrine or bradykinin, being injected simultaneously with prostacyclin, provided faster partial recovery of depressor responses (at 1 h) and promoted some activation of vagal cardiac nerves, despite the effect of indomethacin. Injection of indomethacin did not change the pattern of sympathetic afferent activity. It is suggested that the main derivative of cyclo-oxygenase pathway of arachidonic acid metabolism, prostacyclin, is able to modulate vagal nervous activity at the level of afferent structures in the heart. Prostacyclin may appear a humoral component of cardiogenic depressor reflexes of a vagal nature.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 53–61, January–February, 1996.     

Effect of Inhibition of NO Synthases on Cardiogenic Depressor Reflexes in Different Animal Species

We studied the role of the nitric oxide (NO) system in the realization of cardiogenic depressor reflexes evoked by stimulation of cardiac receptors by veratrine (reproduction of the Bezold–Jarish reflex). Acute experiments were performed on anesthetized dogs and rats: we tested the effects of inhibition of dissimilar isoforms of NO synthase (NOS) and paid special attention to possible species-related differences in realization of the reflex responses. We found that systemic inhibition of NOS by L-nitro-N-arginine (L-NNA, 30 mg/kg, i.v.) significantly decreased the depressor reflex reaction in dogs. Vasomotor dilatatory reactions of the peripheral vessels underwent considerable modifications and in some cases were converted into vasoconstrictory responses. Selective inhibition of neuronal NOS (nNOS) by 7-nitroindazole (7-NI, 25 mg/kg, i.p.) exerted no effect on the development of cardiogenic depressor reflexes in dogs. At the same time, systemic inhibition of NOS in the course of reproduction of cardiogenic depressor reflexes in rats resulted in intensification of depressor responses, while inhibition of nNOS decreased these reactions. Thus, we first demonstrated the role of NO in the realization of cardiogenic depressor reflexes under in vivo conditions and described species-related peculiarities of the involvement of the NO system in the development of these reflexes. We also demonstrated the dependence of formation of cardiogenic depressor reflexes on the predominant involvement of one NOS type or another.     

Vasoconstrictor effects of leukotrienes C4 and D4 in the feline mesenteric vascular bed

The effects of leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD₄) in the feline mesenteric vascular bed were investigated under conditions of controlled blood flow so that changes in perfusion pressure directly reflect changes in vascular resistance. Intra-arterial injections of LTC₄ and LTD₄ (0.3–3.0 μg) increased perfusion pressure in a dose-related fashion. Vasoconstrictor responses to LTC₄ and LTD₄ were similar to norepinephrine (NE) whereas mesenteric vasoconstrictor response to the thromboxane analog, U46619, was markedly greater than were responses to LTC₄ and LTD₄. Meclofenamate in a dose that greatly attenuated the systemic depressor response to arachidonic acid was without effect on vasoconstrictor responses to LTC₄ and LTD₄, NE and U46619 in the mesenteric vascular bed. The present data show that LTC₄ and LTD₄ possess significant vasoconstrictor activity in the feline mesenteric vascular bed. In addition, the present data suggest that products of the cyclooxygenase pathway do not mediate vasoconstrictor responses to LTC₄ and LTD₄ in the intestinal circulation of the cat.     

Effect of phosphocreatine on reflex activity of the brain stem reticular formation

The effects of phosphocreatine (PCr) on startle reflexes, known to be relayed through the brain stem reticular formation (RF), were investigated on chloralose-anesthetized rats. PCr (10^–6 to 10^–3 M) was either applied superficially to the bottom of the fourth ventricle or microinjected into the reticular gigantocellular nucleus. The PCr effect was found to depend on its concentration. At low concentrations (10^–6 to 10^–5 M), PCr markedly facilitated the reflexes; sometimes its application gave rise to additional, later and longer, discharges following startle reflexes, whereas an inhibitory effect predominated at higher PCr concentration (10^–4 to 10^–3 M). Possible mechanisms of PCr action on the studied reflexes are discussed.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 272–278, 1993.     

The effect of leucotriene C4 and D4 on cutaneous blood flow in humans

Using a laser-Doppler-flowmeter the microvascular response to LTC₄ and LTD₄ was measured. Intradermal injection of 1 Ug LTC₄ and LTD₄ caused an increase in the microvascular cutaneous bloodflow. The increase in flow was equal to that caused by histamine in equimolar amounts. Blocking the triple-response did not change the response. The values measured after injection of histamine and leucotrienes were about 10–15 times the values found in undisturbed skin and represents probably a maximally dilated vascular bed. Injection of the leucotrienes caused a slight sensation of pain.     

Organization of Efferent Sympathetic Influences Related to Cardiogenic Depressor Reflexes

In acute experiments on anesthetized dogs under open chest conditions, we studied characteristics of the efferent sympathetic influences on the heart and vessels related to realization of cardiogenic depressor vagus-mediated reflexes. Catheterization of the heart cavities and parallel recording of the mass efferent spike activities in the cardiac and vertebral sympathetic nerves and of the pressure in the aortic ventricle of the heart were used. We found that reflex shifts in the spike activity in the cardiac and vertebral nerves elicited by pharmacological stimulation of the left heart (intracoronary injections of veratrine or adrenaline) and by its nidal immune impairment resulting from injection of a cytotoxic serum demonstrate similar direction (a drop in the frequency of the efferent sympathetic activity). Yet, the dynamics of such inhibitory responses to the influence of the same stimulus and their intensity in one nerve or another and those in one and the same nerve under the influence of different stimuli are considerably dissimilar. Thus, realization of vagus-mediated cardiogenic reflexes is characterized by clear heterogeneity of the efferent sympathetic control of different regions of the cardiovascular system. Such a specificity can provide differential regulation of the heart function and functions of the vascular bed related to different cardiogenic influences (both in the norm and under conditions of formation of an injury nidus in the heart).     

Possible involvement of thromboxane in bronchoconstrictive and hypertensive effects of LTC4 and LTD4 in guinea pigs

The actions of leukotriene (LT) C₄ and D₄ on the systemic arterial pressure and the insufflation pressure in guinea pigs and rabbits were examined. In guinea pigs, 0.3 – 3 nmole/kg of LTC₄ and 0.1 – 1.0 nmole/kg of LTD₄ administrated from left jugular vein caused dose-dependent increase of the airway resistance measured by the Konzett-Rössler method and a triphasic blood pressure response; an initial hypotension, a secondary hypertension and a third long-lasting hypotension. All of the hypertensive phase and 100 – 150% of the increase of the airway resistance by LTC₄ and LTD₄ were inhibited by a selective thromboxane synthetase inhibitor, OKY-1581 (10 mg/kg, i.v.) and only the hypertension was observed. Indomethacin (10 mg/kg, i.p.) also inhibited not only the airway resistance increase, but also the prolonged hypotension by LTC₄ and shortened the duration of the hypotension by LTD₄. It is suggested that thromboxane might be involved in bronchoconstriction and hypertensive effects by LTC₄ and LTD₄ and that hypotensive prostaglandin might be involved in the hypotensive phase after LTC₄ and LTD₄. In rabbits, the increse of the airway resistance by LTC₄ and LTD₄ (upto 100 nmole/kg, i.v.) was negligible and only the hypotension was observed.     

Interaction between leukotriene C4 and histamine in the guinea-pig

Lipoxygenase metabolites have proposed as potential chemical mediators of the bronchial hyperractivity which characterizes asthma (2,6). In addition to the possibility that leukotrienes (LTs) sensitize airways smooth muscle to the contractile actions of other mediators such as histamine (1–3), a number of studies have provided evidence for LT-induced enhancement of bronchoconstriction by a vagal dependent mechanism (4–6). In the present study the effects of exposure of the airway to LTC₄ on subsequent responsiveness to histamine have been investigated in both and experiments. LTC₄, in a concentration eliciting threshold contractile responses of the isolated trachea (1.7 nM), had no effect on either the EC₅₀ or maximal contractile response to histamine. At a concentration eliciting an approximately EC₅₀ contractile response, LTC₄ (10 nM) shifted the histamine concentration-response curve rightwards altering the maximum response. In anaesthetized, mechanically ventilated guinea pigs LTC₄ (0.1–0.4 nMole/kg, i.v.) injected 20 s beforehand, failed to alter histamine (9–36 nMole/kg, i.v.)-induced bronchoconstriction whereas, under the same conditions, LTD₄ (0.05–0.2 nMole/kg, i.v.) dose-dependently enhanced histamine-induced bronchoconstriction. On the other hand, LTC₄ or LTD₄ (16 uM, 30 s) aerosols potentiated histamine (9.36 nMole/kg, i.v.) in a concentration-dependent manner (Table). Both LTC₄ and LTD₄ aerosols enahance airway reactivity to histamine whereas only LTD₄ has this action when administered intravenously. Neither LTC₄ nor LTD₄ (6) enhances the contractile effects of histamine on isolated airways smooth muscle. It is concluded that the broncho-constriction enhancing action of these leukotrienes may be indirectly mediated.     

Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonim of leukotriene (LT) C₄-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cycloocygenase inhibitor indomethacin, NDGA (30 μM) as well as idomethacin (5 μM) inhibited LTC₄-iduced contraction. But in the presence of indomethacin NDGA was ineffective to inhibit the LTC₄ response, whereas two other lipoxygenase inhibitors, phenidone (3–30 μM) and 5,8,11,14-eicostatetraynoic acid (ETYA, 10 μM), markedly inhibited it. The antagonist action of an LTD₄ receptor antagonist FPL55712 against LTC₄-induced contractions was significantly reduced by NDGA (10–30 μM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect n the LTC₄ antagonism in the presence of indomethacin, but 0.3 μM phenidone and 1 μM ETYA which did not inhibit the LTC₄ response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC₄, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LCT₄ response was not inhibited by indomethacin, 0.3 μM phenidone and 1 μM ETYA. In the presence of a γ-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC₄ antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 μM histamine- and 30 μM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H₁-blocker pyrilamine. These results suggest that some putative nonprostanoids are involved in LTC₄-induced contractions of guinea-pig trachea and which regulate the effects of LTD₄ antagonism and β-adrenoceptor activation.     

Retinoid-induced inhibition of bosinophil LTC4 production

Naturally occuring and synthetic retinoids demonstrate a marked antiinflammatory effect when employed in such disorders as acne and psoriastis. This effect may result in part from their inhibition of release of potent mediators (e.g. eicosanoids) by inflammatory cells. In this study, we examined the effect of eight retinoids (tretinoin, isotretinoin, retinol, retinal, acitretin, retinyl palmitate, etretinate, Ro 15–0778) on the release of leukotriene (LT)C₄, an important lipid mediator generated by eosinophils. Tretinoin, isotretinoin, retinol, retinal, and acitretin at 10⁻⁵ M or 10⁻⁴ M concentrations inhibited LTC₄ release by A23187-stimulated horse eonsinophils in vitro; 10⁻⁴ M retinyl palmitate was also inhibitory. However, 10⁻⁵ M etretinate augmented A23187-induced LTC₄ release, and the arotinoid Ro 15–0778 had no effect on LTC₄ production. These data suggest that selected retinoids may have potential use in the reduction of LTC₄ generation by eosinophils. This inhibition could be beneficial in the theraphy of such diseases as bronchial asthma in which release of LTC₄ may be involved in the inflammtory process.     

Effects of LTC4 and BW 755C,a blocker of its synthesis,on contraction in smooth muscle and operation of voltage-dependent calcium channels in nerve cells

The effects were investigated of LTC₄, a synthetic leukotriene, and BW 755C, a blocker of LTC₄ biosynthesis, on the operation of Ca channels at the cell membrane and on contraction of muscle fibers using intracellular dialysis and voltage clamping at the membrane of isolated nerve cells and by recording spontaneous contraction of the uterus in white rats at advanced stages of pregnancy. It was found that 1·10^–7 M LTC₄ stimulates the contraction of the uterus without altering its response to oxytocin application. The same concentration of LTC₄ was found to increase calcium conductance by 60±27%. At the same time, a 25±6 mV shift in peak current-voltage relationship along the voltage axis toward negative values was recorded for calcium current. BW 755C, a blocker of the key enzyme in the lipoxygenase metabolic pathway of arachidonic acid, exerts an action similar to leukotriene on calcium conductance, although brief contraction of the uterus is rapidly replaced by complete inhibition of this activity.Institute of Bioorganic Chemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 24–31, January–February, 1989.     

Comparative study of the pulmonary effects of intravenous leukotrienes and other bronchoconstrictors in anaesthetized guinea pig

Pulmonary responses to intravenous leukotrienes C₄, D₄ and E₄ administered as a bolus injection and by continuous infusion were studied in anesthetized guinea pigs. LTD₄, LTC₄ and LTE₄ (respective ED₅₀ of 0.21 ± .1, 0.64 ± .2 and 2.0 ± .1 μg kg⁻¹) produced dose-dependent increases in insufflation pressure when given as a bolus injection to anesthetized guinea pigs (Konzett-Rössler). Bronchoconstriction was antagonized by FPL-55712 (50–200 μg kg⁻¹), and indomethacin (50–200 μg kg⁻¹) but was not significantly altered by mepyramine (1.0 mg kg⁻¹), methysergide (0.1 mg kg⁻¹), intal (10 mg kg⁻¹) mepacrine (5 mg kg⁻¹) or dexamethasone (10 mg kg⁻¹). The beta adrenoceptor blocker, timolol (5 μg kg⁻¹) produced a significantly greater potentiation of the responses to the leukotrienes than to arachidonic acid, histamine and acetylcholine. Responses to bolus injection of LTE₄ but not LTD₄ or LTC₄ were partially antagonized by atropine (100 μg kg⁻¹) and bilateral vagotomy. In experiments of a different design, continuous infusion of LTD₄ and LTE₄ (2.8–3.2 μg kg⁻¹ min⁻¹) into indomethacin-treated animals produced slowly developing increases in pulmonary resistance and decreases in compliance. The increase in resistance produced by LTE₄ and LTD₄ was partly reversed by intravenous FPL-55712 (1.0 mg kg⁻¹) and atropine (100 μg kg⁻¹) but was almost completely reversed by FPL-55712 (3 – 10 mg kg⁻¹). These findings indicate that leukotrienes can produce bronchoconstriction in guinea pigs through cyclooxygenase-dependent and cyclooxygenase independent mechanisms both of which are blocked by FPL-55712. Cholinergic mechanisms are involved in the mediation of part of the response to bolus injection of LTE₄ as well as a small part of the initial response to continuous infusion of LTD₄ and LTE₄. Intrinsic beta adrenoceptor activation serves to down modulate responses to the leukotrienes to a greater extent than responses to arachidonic acid, histamine and acetylcholine.     

Arachidonic Acid and Leukotriene C4: Role in Transient Cerebral Ischemia of Gerbils

Accumulation of arachidonic acid (AA) is greatest in brain regions most sensitive to transient ischemia. Free AA released after ischemia is either: 1) reincorporated into the membrane phospholipids, or 2) oxidized during reperfusion by lipoxygenases and cyclooxygenases, producing leukotrienes (LT), prostaglandins, thromboxanes and oxygen radicals. AA, its metabolite LTC₄ and lipid peroxides (generated during AA metabolism) have been implicated in the blood-brain barrier (BBB) dysfunction, edema and neuronal death after ischemia/reperfusion. This report describes the time course of AA release, LTC₄ accumulation and association with the physiological outcome during transient cerebral ischemia of gerbils. Significant amount of AA was detected immediately after 10 min ischemia (0 min reperfusion) which returned to sham levels within 30 min reperfusion. A later release of AA occurred after 1 d. LTC₄ levels were elevated at 0–6 h and 1 d after ischemia. Increased lipid peroxidation due to AA metabolism was observed between 2–6 h. BBB dysfunction occurred at 6 h. Significant edema developed at 1 and 2 d after ischemia and reached maximum at 3 d. Ischemia resulted in ~80% neuronal death in the CA₁ hippocampal region. Pretreatment with a 5-lipoxygenase inhibitor, AA861 resulted in significant attenuation of LTC₄ levels (Baskaya et al. 1996. J. Neurosurg. 85:112–116) and CA₁ neuronal death. Accumulation of AA and LTC₄, together with highly reactive oxygen radicals and lipid peroxides, may alter membrane permeability, resulting in BBB dysfunction, edema and ultimately to neuronal death.     

Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): Nonperoxidative purine and pyrimidine nucleotide depletion

Hydrogen peroxide (H₂O₂) overload may contribute to cardiac ischemia-reperfusion injury. We report utilization of a previously described cardiomyocyte model (J. Cell. Physiol., 149:347, 1991) to assess the effect of H₂O₂-induced oxidative stress on heart-muscle purine and pyrimidine nucleotides and high-energy phosphates (ATP, phosphocreatine). Oxidative stress induced by bolus H₂O₂ elicited the loss of cardiomyocyte purine and pyrimidine nucleotides, leading to eventual de-energization upon total ATP and phosphocreatine depletion. The rate and extent of ATP and phosphocreatine loss were dependent on the degree of oxidative stress within the range of 50 μM to 1.0 mM H₂O₂. At the highest H₂O₂ concentration, 5 min was sufficient to elicit appreciable cardiomyocyte highenergy phosphate loss, the extent of which could be limited by prompt elimination of H₂O₂ from the culture medium. Only H₂O₂ dismutation completely prevented ATP loss during H₂O₂-induced oxidative stress, whereas various freeradical scavengers and metal chelators afforded no significant ATP preservation. Exogenously-supplied catabolic substrates and glycolytic or tricarboxylic acidcycle intermediates did not ameliorate the observed ATP and phosphocreatine depletion, suggesting that cardiomyocyte de-energization during H₂O₂-induced oxidative stress reflected defects in substrate utilization/energy conservation. Compromise of cardiomyocyte nucleotide and phosphocreatine pools during H₂O₂-induced oxidative stress was completely dissociated from membrane peroxidative damage and maintenance of cell integrity. Cardiomyocyte de-energization in response to H₂O₂ overload may constitute a distinct nonperoxidative mode of injury by which cardiomyocyte energy balance could be chronically compromised in the post-ischemic heart. © 1993 Wiley-Liss, Inc.     

Leukotriene-C4 Synthase,a Critical Enzyme in the Activation of Store-independent Orai1/Orai3 Channels,Is Required for Neointimal Hyperplasia

Leukotriene-C₄ synthase (LTC₄S) generates LTC₄ from arachidonic acid metabolism. LTC₄ is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC₄ was also a cytosolic second messenger that activated store-independent LTC₄-regulated Ca²⁺ (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC₄S in neointima formation remains unknown. Here we show that LTC₄S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC₄S protein expression in VSMCs. Inhibition of LTC₄S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC₄S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC₄S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC₄S or Orai3 was prevented by shRNA. We conclude that LTC₄S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.     

Interaction of leukotriene C4 and chinese hamster lung fibroblasts (V79A03 cells). 2. Subcellular distribution of binding and unlikely role of glutathione-S-transferase

It was reported previously that radiation-induced cytotoxicity in V79A03 (V79) cells was attenuated by pretreatment of cells with leukotriene C₄ (LTC₄), leading us to determine that V79 cells possessed specific binding sites, with characteristics of receptors, for LTC₄ (see the preceding, companion communication). Additional studies were conducted to determine the subcellular distribution and the chemical nature of the LTC₄ binding site in V79 cells. Trypsin treatment of cells before LTC, binding assays resulted in a 74% reduction in high-affinity binding. In tests to examine the subcellular location of LTC₄ binding, plasma membrane and nuclear fractions were obtained from V79 cells. In contrast to Scatchard analyses of LTC₄ binding to intact cells which were curvilinear, Scatchard analyses of nuclear and plasma membrane fractions were linear, indicative of the presence in these cellular substituents of low and high-affinity binding, respectively. To examine the nature of the high-affinity LTC₄ binding sites, intact V79 cells were photolyzed with [³H]-LTC₄ rendered photoactive by preincubation with N-hydroxysuccinimidyl-4-azidobenzoate. The cell-bound radioactivity migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of approaximately 40 kdal. Five different commercial preparations of glutathione-S-transferase (GST), which has been implicated as a source of LTC₄ “specific binding” in other cells, migrated in the same SDS_PAGE system with an apparent molecular weigth of 20–24 kdal. Furthermore, preincubations of V79 cells with three antisera generated against GST had minimal effects upon subsequent LTC₄ binding to intact cells. These data, suggest that the radioprotective effect of LTC₄ upon V79 cells may be attributable to a receptor-mediated phenomenon which appears distinct from leukotriene binding to GST.     

Late long-lasting discharges in hind limb motor nerves and their connection with the locomotor rhythm in thalamic immobilized cats

Effects of stimulation of flexor reflex afferents were studied in decerebrate immobilized cats. Stimulation of ipsilateral afferents evoked late long-lasting discharges in the nerves to the flexors, whereas stimulation of contralateral afferents led to similar discharges in nerves to both extensors and flexors. Compared with spinal animals, early segmental reflexes in thalamic cats were tonically depressed. Similar tonic inhibition of segmental reflexes took place in spinal animals after injection of dopa. Segmental reflexes were clearly modulated during late or rhythmic discharges. The possible central mechanisms of these changes in the segmental reflexes are discussed on the basis of data in the literature.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 2, pp. 137–145, March–April, 1979.     

Role of NO in coronary and systemic vasodilation following cardiogenic reflexes

In acute experiments on anesthetized dogs under closed-chest conditions, we used the technique of double lumen catheterization of coronary vessels and peripheral vessel bed. We studied the role of endothelium-dependent relaxing factor/nitric oxide (EDRF/NO) in the development of parasympathetic coronary vasodilation after excitation of cardiac receptors. Under conditions of pharmacological stimulation of cardiac receptors of the left ventricle and short-lasting episodes of local myocardial ischemia, we also examined the effects of inhibition of NO synthesis on the development of cardiogenic depressor reflexes (hypotension and peripheral vasodilation). It was found that the reflex coronary dilatation following excitation of the cardiac (left ventricular) receptors significantly decreased after systemic NO synthase inhibition. Thus, NO production is one of the effector mechanisms of the development of coronary vessel dilatation; this conclusion is confirmed by changes in the dilatation level after blockade of this process with L-NNA (nitro-ω-L-arginine). We pioneered in demonstrating that after the blockade of NO synthesis peripheral vessel vasodilation decreases or disappeas altogether when cardiogenic reflexes are realized following pharmacological excitation of cardiac receptors with veratrine or catecholamine injections, and vasoconstrictor responses evoked by myocardial ischemia are significantly intensified. It is suggested that the influences of NO-dependent mechanisms exert a dual effect on sympathic control-mediated peripheral vasodilation during cardiogenic reflexes. Such mechanisms reduce central sympathetic tone and/or concurrently provide peripheral inhibition of neural sympathetic influences; in the latter case, NO-dependent cardiogenic reflexes play a crucial role in compensatory reactions after an injury to the heart.