Behavioural responses of salmonid fry to low amino acid concentrations

The behaviour of start feed brown trout, Salmo trutla , and Atlantic salmon, Salmo salar , fry was observed in response to 5, 50 and 500 nM concentrations of L-alanine, _L-proline, L-arginine and glycine. In addition, salmon fry were tested with dilute concentrations of shrimp extract. Five behaviour patterns (snap, yawn, dart, twitch movements and active swimming) were shown in response to all amino acid and shrimp extract concentrations. Snapping, darting and active swimming increased in both species as a function of amino acid concentration, and in the salmon fry as a function of increasing shrimp extract concentration. Otherwise, the salmon showed twice as much yawning and more twitch movements than the trout, but the trout showed more active swimming than the salmon. Both species showed an increase in activity in response to L-proline at 5 nM, and the salmon also responded to L-arginine and glycine at this concentration. Both species first responded to L-alanine at 50 nM, but the trout did not respond to glycine until a concentration of 500 nM was presented. The salmon fry responded to shrimp extract at c. 10 ¹⁴g l^-1, but no differences in their activity were observed in response to concentrations between c. 10^-12 and 10^-6gl^-1.     

Endothelium-derived hyperpolarizing factor in coronary microcirculation: responses to arachidonic acid

In coronary resistance vessels, endothelium-derived hyperpolarizing factor (EDHF) plays an important role in endothelium-dependent vasodilation. EDHF has been proposed to be formed through cytochrome P-450 monooxygenase metabolism of arachidonic acid (AA). Our hypothesis was that AA-induced coronary microvascular dilation is mediated in part through a cytochrome P-450 pathway. The canine coronary microcirculation was studied in vivo (beating heart preparation) and in vitro (isolated microvessels). Nitric oxide synthase (NOS) (N(omega)-nitro-L-arginine, 100 microM) and cyclooxygenase (indomethacin, 10 microM) or cytochrome P-450 (clotrimazole, 2 microM) inhibition did not alter AA-induced dilation. However, when a Ca(2+)-activated K(+) channel channel or cytochrome P-450 antagonist was used in combination with NOS and cyclooxygenase inhibitors, AA-induced dilation was attenuated. We also show a negative feedback by NO on NOS-cyclooxygenase-resistant AA-induced dilation. We conclude that AA-induced dilation is attenuated by cytochrome P-450 inhibitors, but only when combined with inhibitors of cyclooxygenase and NOS. Therefore, redundant pathways appear to mediate the AA response in the canine coronary microcirculation.     

Pulmonary microvascular responses to arachidonic acid in isolated perfused guinea pig lung

We examined the effects of arachidonic acid (AA) on pulmonary hemodynamics and fluid balance in Ringer- and blood-perfused guinea pig lungs during constant-flow conditions. Mean pulmonary arterial (Ppa), venous (Pv), and capillary pressures (Pcap, estimated by the double-occlusion method) were measured, and arterial (Ra) and venous resistances (Rv) were calculated. Bolus AA injection (500 micrograms) caused transient increases (peak response 1 min post-AA) in Ppa, Pcap, and Rv without affecting Ra in both Ringer- and blood-perfused lungs. The response was sustained in blood-perfused lungs. AA had no effect on the capillary filtration coefficient in either Ringer- or blood-perfused lungs. AA stimulated the release of thromboxane B2 and 6-ketoprostaglandin F1 alpha in both Ringer- and blood-perfused lungs, but the responses were sustained only in the blood-perfused lungs. Meclofenamate (1.5 X 10(-4) M), a cyclooxygenase inhibitor, abolished the AA-induced pulmonary hemodynamic responses in both Ringer- and blood-perfused lungs, whereas U-60257 (10 microM), a lipoxygenase inhibitor, attenuated the response only in the blood-perfused lungs. In conclusion, AA does not alter pulmonary vascular permeability to water in either Ringer- or blood-perfused lungs. AA mediates pulmonary venoconstriction and thus contributes to the rise in Pcap. The venoconstriction results from the generation of cyclooxygenase-derived metabolites from lung parenchymal cells and blood-formed elements. Lipoxygenase metabolites may also contribute to the vasoconstriction in the blood-perfused lungs.     

Effects of OKY 1581 on bronchoconstrictor responses to arachidonic acid and PGH2

The influence of OKY 1581, a thromboxane synthase inhibitor, on airway responses to arachidonic acid and endoperoxide, [prostaglandin (PG) H2], were investigated in anesthetized, paralyzed, mechanically ventilated cats. Intravenous injections of arachidonic acid and PGH2 caused dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic and static compliance. OKY 1581 significantly decreased airway responses to arachidonic acid but not to PGH2. Sodium meclofenamate, a cyclooxygenase inhibitor, abolished airway responses to arachidonic acid but had no effect on airway responses to PGH2. OKY 1581 or meclofenamate has no effect on airway responses to PGF2 alpha, PGD2, or U 46619, a thromboxane mimic. In microsomal fractions from the lung, OKY 1581 inhibited thromboxane formation without decreasing prostacyclin synthesis or cyclooxygenase activity. These studies show that OKY 1581 is a selective thromboxane synthesis inhibitor in the cat lung and suggest that a substantial part of the bronchoconstrictor response to arachidonic acid is due to thromboxane A2 formation. Moreover, the present data suggest that airway responses to endogenously released and exogenous PGH2 are mediated differently and that a significant part of the response to exogenous PGH2 may be due to activation of an endoperoxide/thromboxane receptor, since responses to PGH2 are blocked by the thromboxane receptor antagonist SQ 29548.     

Protein kinases in plant responses to drought,salt, and cold stress

Protein kinases are major players in various signal transduction pathways. Understanding the molecular mechanisms behind plant responses to biotic and abiotic stresses has become critical for developing and breeding climate-resilient crops. In this review,we summarize recent progress on understanding plant drought, salt, and cold stress responses, with a focus on signal perception and transduction by different protein kinases, especially sucrose nonfermenting1(SNF1)-related protein kinases(Sn RKs),mitogen-activated protein kinase(MAPK) cascades,calcium-dependent protein kinases(CDPKs/CPKs),and receptor-like kinases(RLKs). We also discuss future challenges in these research fields.     

Protein kinase C subspecies in adult rat hippocampal synaptosomes. Activation by diacylglycerol and arachidonic acid

Synaptosomes isolated from the adult rat hippocampus contain the alpha- and beta-subspecies of protein kinase C (PKC), but not the gamma-subspecies which is abundantly expressed in the pyramidal cells in this brain region. Although the gamma-subspecies is known to respond significantly to free arachidonic acid, it is found that both the alpha- and beta-subspecies are also activated dramatically by arachidonic acid in synergistic action with diacylglycerol. Oleic, linoleic, and linolenic acids are all active. It is possible that unsaturated fatty acids may take part in the activation of alpha- and beta-subspecies of PKC which are present in the presynaptic nerve endings terminating at the hippocampal pyramidal cells.     

Protein kinase C translocation in human blood platelets

Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 microM concentrations did not induce redistribution of platelet PKC. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10-100 microM) but not the 5-HT1A or 5-HT1B agonists, (+/-) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT2 receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT2 receptors.     

Protein kinases from Dictyostelium discoideum with similarity to LIM kinases

We cloned a protein kinase (DdKinY) from Dictyostelium discoideum by low stringency hybridization using the catalytic domain from DdKinX [B.W. Wetterauer et al., Biochim. Biophys. Acta 1265 (1995) 97-101] as a probe. Both kinases have low sequence similarity to other protein kinases in the databases. However, phylogenetic analysis showed that both kinases cluster with vertebrate LIM kinases due to homology within the catalytic domain.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid systhesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3,6, or 30μg/ml) was placed under cranial windows in newborn pigs that been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentration, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dosedependent increases of PGE₂ in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF_1α and TXB₂ only increased at the highest concentration of arachidonic acid (30 μg/ml). Cerebral ischemia did not decrease the conservation of any concentration of arachidonic acid to PGE₂, 6-keto-PGF_1α, or TXB₂. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid synthesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3, 6, or 30 micrograms/ml) was placed under cranial windows in newborn pigs that had been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentrations, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dose-dependent increases of PGE2 in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF1 alpha and TXB2 only increased at the highest concentration of arachidonic acid (30 micrograms/ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto-PGF1 alpha, or TXB2. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Biophysical responses of red cell-membrane systems to very low concentrations of inorganic mercury

Changes in red blood cell size, deformability, and osmotic fragility are indicators of altered condition and/or altered regulatory processes at the whole cell and membrane levels. An agent, such as HgCl2, that brings about specific changes of this kind can therefore serve as a selective probe of such cell condition and regulatory state. Conversely, for a health-threatening agent "active" in this way, the cell-membrane responses serve to clarify the more fundamental bases of its toxicity, as well as to permit identification and characterization of its early and low-level actions on living systems. Taking advantage of recent advances in the technique of "resistive pulse spectroscopy," we present a coordinated study of these three interrelated biophysical properties for the interactions of HgCl2 with human red cells. We thereby are able to extend previous studies of this kind into domains of shorter time (instantaneous exposures), lower level exposures (down to 10(-9) M, well below the level of acute human toxicity), as well as to additional kinds of responses (e.g., "dynamic osmotic hemolysis"). For conditions ranging from 10(-4) to 10(-9) M in HgCl2, for instantaneous to 90-min-incubated exposures, for medium osmolarities from 120 to 300, the matrix of observed cell responses includes relative swelling as well as shrinkage, changes in deformability, and both enhancement of and protection against osmotic hemolysis. Some unexpected short-term effects of time and temperature of storage of blood cell stock samples, with respect to increasing and decreasing osmotic fragility, are also reported. These apparently disparate results are interpreted in terms of mercury interactions with cell and membrane SH groups, and a reasonable rationale is presented for most of the responses in terms of disruption of passive and active Na+-K+, gradient controls, plus interactions with cellular proteins.     

Biophysical responses of red cell-membrane systems to very low concentrations of inorganic mercury

Changes in red blood cellsize, deformability, andosmotic fragility are indicators of altered condition and/or altered regulatory processes at the whole cell and membrane levels. An agent, such as HgCl₂, that brings about specific changes of this kind can therefore serve as a selective probe of such cell condition and regulatory state. Conversely, for a health-threatening agent “active” in this way, the cell-membrane responses serve to clarify the more fundamental bases of its toxicity, as well as to permit identification and characterization of its early and low-level actions on living systems. Taking advantage of recent advances in the technique of “resistive pulse spectroscopy,” we present a coordinated study of these three interrelated biophysical properties for the interactions of HgCl₂ with human red cells. We thereby are able to extend previous studies of this kind into domains of shorter time (instantaneous exposures), lower level exposures (down to 10⁻⁹ M, well below the level of acute human toxicity), as well as to additional kinds of responses (e.g., “dynamic osmotic hemolysis”). For conditions ranging from 10⁻⁴ to 10⁻⁹ M in HgCl₂, for instantaneous to 90-min-incubated exposures, for medium osmolarities from 120 to 300, the matrix of observed cell responses includes relative swelling as well as shrinkage, changes in deformability, and both enhancement of and protection against osmotic hemolysis. Some unexpected short-term effects of time and temperature of storage of blood cell stock samples, with respect to increasing and decreasing osmotic fragility, are also reported. These apparently disparate results are interpreted in terms of mercury interactions with cell and membrane SH groups, and a reasonable rationale is presented for most of the responses in terms of disruption of passive and active Na⁺−K⁺, gradient controls, plus interactions with cellular proteins.     

Influence of OKY-1581 on responses to arachidonic acid in the feline pulmonary vascular bed

The effects of OKY-1581, a thromboxane synthesis inhibitor, on pulmonary vascular responses to arachidonic acid (AA) were investigated under baseline and elevated tone conditions in the intact chest cat. Under conditions of controlled blood flow at baseline tone, intralobar injections of AA increased lobar arterial pressure in a dose-related manner. These pressor responses were reduced by OKY-1581, and a small vasodilator response was unmasked. The administration of indomethacin to these same animals abolished all responses to AA. When baseline tone in the pulmonary vascular bed was elevated by infusion of U46619, intralobar injections of AA caused a biphasic change in lobar arterial pressure characterized by an initial increase followed by a secondary fall in pressure. Treatment with OKY-1581 attenuated the pressor component of the response and enhanced the depressor component of the response. All responses to AA at elevated tone were also blocked by indomethacin. Pressor responses to intralobar injections of U46619 were not altered by OKY-1581 or indomethacin and were similar under baseline and high pulmonary vascular tone conditions. The results of this study suggest that the pulmonary pressor response to AA in the cat is dependent in large part on the formation of TXA2 and also suggest that TXA2, PGI2, and vasoconstrictor prostaglandins (PGF2 alpha, PGD2, PGE2) are formed from AA in the cat lung.     

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.