3-phenyllactic acid production by free-whole-cells of Lactobacillus crustorum in batch and continuous fermentation systems.

AIM: 3-Phenyllactic acid (3-PLA) has been widely used in food and material industries. Three Lactobacillus crustorum strains have shown greater 3-PLA production ability in our previous study. The objectives of this study were to further improve 3-PLA yields in batch and continuous fermentation systems using of free-whole-cells of the three L. crustorum strains. MATERIALS AND RESULTS: The fermentation conditions of free-whole-cells of the three L. crustorum strains for 3-PLA production were optimized. Among these strains, L. crustorum NWAFU 1078 showed excellent reusability and significantly (P < 0·05) greater 3-PLA production ability than the other strains after 10th recycle. The strain possesses three l-lactate dehydrogenase and three d-lactate dehydrogenase catalysing 3-PLA production from phenylpyruvic acid (PPA). Under the optimal conditions, the strain produced 15·2 mmol l-1 3-PLA (76% PPA conversion rate) in a batch fermentation system and 6·5 mmol l-1  h-1 3-PLA (55% PPA conversion rate) in a continuous fermentation system using a 0·6 dilution rate. CONCLUSIONS: Free-whole-cells of L. crustorum NWAFU 1078 showed excellent reusability and higher 3-PLA yields under optimal biotransformation conditions in both batch and continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the possibility to use the free-whole-cells of L. crustorum NWAFU 1078 as a biocatalyst for effective production of 3-PLA.

Lactobacillus coryniformis subsp. torquens inhibits bone loss in obese mice via modification of the gut microbiota.

High-fat diet (HFD)-induced obesity results in bone loss associated with an imbalanced gut microbiota and altered immune status. Probiotics are live microorganisms that are beneficial to the host and are important in maintaining bone health and gut homeostasis. In this study, the probiotic Lactobacillus coryniformis subsp. torquens (T3L) was isolated from traditional yak milk cheese produced in Lhasa and showed distinct acid and bile salt resistance as potential probiotics. Our data indicated that T3L not only reversed HFD-induced gut dysbiosis, as indicated by decreased Firmicutes-to-Bacteroidetes ratios but also reduced bone loss. The anti-obesity, microbiome-modulating, and bone-protective effects were transmissible via horizontal faeces transfer from T3L-treated mice to HFD-fed mice. The protective effects of T3L on bone mass were associated with regulatory T (Treg) cell-mediated inhibition of osteoclast differentiation. Our data indicate that T3L is a regulator of the gut microbiota and bone homeostasis in an animal model.

The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects.

BACKGROUND AND PURPOSE: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. EXPERIMENTAL APPROACH: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. KEY RESULTS: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. CONCLUSIONS: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.

Expression of monocyte chemotactic protein-3 mRNA in rat vascular smooth muscle cells and in carotid artery after balloon angioplasty.

Monocyte chemotactic protein-3 (MCP-3) is a CC chemokine that functions in chemoattraction and activation of monocytes, T lymphocytes, eosinophils, basophils, natural killer cells and dendritic cells. The activation of the target cells by MCP-3 is via specific chemokine receptors CCR2 and CCR3, of which CCR2 is shared with MCP-1. MCP-1 and CCR2 have been implicated in vascular diseases including atherosclerosis and restenosis, that are known to be involved in inflammation (accumulation of T lymphocytes and monocytes) and smooth muscle cell (SMC) activation (proliferation, migration and matrix deposition). To investigate a potential role of MCP-3 in vascular injury, the present work examined its mRNA expression in rat aortic SMCs stimulated with various inflammatory stimuli including LPS, TNF-alpha, IL-1beta, IFN-gamma and TGF-beta. A time- and concentration-dependant induction of MCP-3 mRNA in SMCs was observed by means of Northern analysis. A strikingly similar expression profile was observed for MCP-3 and MCP-1 mRNA in SMCs. Furthermore, MCP-3 mRNA expression was induced in rat carotid artery after balloon angioplasty. A significant induction in MCP-3 mRNA was observed in the carotid artery at 6 h (41-fold increase over control, P<0.001), 1 day (13-fold increase, P<0.001) and 3 days (6-fold increase, P<0.01) after balloon angioplasty as quantitated by reverse transcription and polymerase chain reaction. These data provide evidence for the cytokine-induced expression of MCP-3 in SMCs and in carotid artery after balloon angioplasty, suggesting a potential role of MCP-3 in the pathogenesis of restenosis and atherosclerosis.

Effects of protein deficiency on the metabolism of arachidonic acid by rat pleural polymorphonuclear leukocytes.

The effects of protein deficiency on the biosynthesis of metabolites of arachidonic acid by rat pleural polymorphonuclear leukocytes stimulated with calcium ionophore were investigated. The major products of metabolism by lipoxygenase in these cells were leukotriene B4 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas the major cyclooxygenase products were thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. At high substrate concentrations (100 microM), the formation of all products by polymorphonuclear leukocytes was lower for protein-deficient rats than for controls. Similar results were obtained when products synthesized from endogenous substrate were measured, except that there was no change in the amount of 5-hydroxy-6,8,11,14-eicosatetraenoic acid formed. The biosynthesis of prostaglandins E2 and F2 alpha by homogenates of rat kidney medulla was reduced as a result of protein deficiency. Acetylsalicylic acid inhibited the formation of cyclooxygenase products and stimulated the formation of lipoxygenase products by polymorphonuclear leukocytes. Protein deficiency did not alter the effects of acetylsalicylic acid on the biosynthesis of these products, although at any given concentration the amounts of products formed were less with protein-deficient rats than with rats fed control diets.

Carvedilol-liposome interaction: evidence for strong association with the hydrophobic region of the lipid bilayers.

Carvedilol (Kredex, Coreg) is a multiple action antihypertensive drug that has been shown to protect cell membranes from lipid peroxidative damages. In this study the physical and structural effects of carvedilol on lipid bilayers are investigated by fluorescence techniques, differential scanning calorimetry and other physical methods. Carvedilol binds to liposomal membranes (9:1 DMPC:DMPG) strongly with an apparent binding constant on the order of 10(4) M-1 in PBS (pH 7.4). The characteristic changes in its intrinsic fluorescence properties when bound to liposomes suggest that this compound is situated in a non-polar environment. The Stern-Volmer and bimolecular quenching constants, determined using nitrate as the fluorescence quencher, for the free and bound carvedilol indicate that the carbazole moiety is at a depth of > 11 A in the lipid bilayer. Fluorescence anisotropy measurements show that, unlike the membrane probes DPH and TMA-DPH, carvedilol is relatively mobile, and does not have a rigidly-defined molecular orientation in the bilayers. Differential scanning calorimetry results indicate that carvedilol is an effective membrane "fluidizer' as it dose-dependently lowers the gel to liquid crystalline transition temperature and broadens the endothermic transition. Comparative studies of interactions of carbazole, 4-OH carbazole and carvedilol with the model liposomal membranes reveal a possible role of membrane-partitioning in their antioxidant efficacy. These findings are discussed in perspective with the membrane biophysical properties of different classes of therapeutic significant lipid antioxidants in mind.

Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

Apoptosis: a potential target for discovering novel therapies for cardiovascular diseases.

The realization that apoptosis is genetically programmed raises the exciting prospect that modulating apoptosis may provide novel approaches for treatment of cardiovascular diseases in which apoptosis has been demonstrated. Low molecular weight inhibitors of caspases and mitogen-activated protein kinases have been evaluated, with promising results in a variety of cardiovascular apoptotic models.

Apoptosis and congestive heart failure.

Congestive heart failure (CHF) is the final clinical manifestation of a variety of cardiac (myopathies), coronary (atherosclerosis), and systemic diseases (diabetes, hypertension). Regardless of the origin of the cardiac insult, left ventricular dysfunction resulting in decreased cardiac output elicits a series of adaptational processes that attempt to compensate for some of the decrement in myocardial function. One of the key manifestations of these compensatory processes is cardiac hypertrophy, which is characterized by a marked increase in myocyte size and an increase in contractile proteins. The benefits resulting from these compensatory adaptational mechanisms, however, are only transient, and within a period of months to years, the changes induced in the myocardium fail to sustain cardiac output at a level that is sufficient to meet the demands of the body; subsequently, physical performance is impaired. Typically, progressive dilation and thinning of the left ventricle occur along with progression of CHF. The mechanisms responsible for the thinning of ventricular tissue and loss of left ventricular mass are poorly understood; traditionally, such loss has been attributed to tissue necrosis based on the morphologic observation of dead cardiac myocytes. Very recently, there have been data suggesting that apoptosis, a form of programmed cell death (PCD), occurs in the heart and may be responsible, at least in part, for the progression of CHF and the chronic loss of left ventricular function and mass. Evidence for a role of apoptosis/PCD in the progression of heart failure has been obtained from a variety of observations, including in vitro studies of cardiac myocytes in culture, experimental animal models of cardiac injury, and cardiac tissue obtained from patients with CHF. Thus, apoptosis/PCD may be a critical mechanism involved in the progressive loss of cardiac myocytes, which ultimately results in end-stage heart failure. In this brief review, the evidence for apoptosis/PCD in cardiac myocytes is presented and its potential role in the progression of CHF is analyzed. In particular, the genomic basis for apoptosis in cardiac myocytes is explored, and its relevance to the identification of novel targets for future pharmacological interventions is discussed. (Trends Cardiovasc Med 1997;7:249-255). © 1997, Elsevier Science Inc.

Enhanced leucocyte adhesion to interleukin-1 beta stimulated vascular smooth muscle cells is mainly through intercellular adhesion molecule-1.

OBJECTIVE: The aim was to investigate whether interleukin-1 beta (IL-1 beta) plays a role in modulating the adhesion of monocytes and neutrophils to vascular smooth muscle cells, and to identify what molecules on these cells may be involved in the adhesion. METHODS: Rat aortic smooth muscle cells were challenged with IL-1 beta and tested for adhesion of prelabelled monocytes and neutrophils. Northern analysis, reverse transcription/polymerase chain reaction (RT/PCR), and immunocytochemical staining were used to measure the changes of intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules in response to IL-1 beta stimulation. Neutralising antibody against ICAM-1 was used to demonstrate a role of ICAM-1 in this IL-1 beta induced adhesion. RESULTS: IL-1 beta induced the adhesion of monocytes and neutrophils to aortic smooth muscle cells in a concentration and time dependent manner. IL-1 beta-induced adhesion was inhibited by preincubation of the cells with an IL-1 receptor antagonist (IL-1ra). Northern analysis and RT/PCR showed that ICAM-1 mRNA represents a predominant adhesion molecule induced by IL-1 beta, and that the expression of ICAM-1 mRNA precedes and parallels the induced adhesion profiles of aortic smooth muscle cells for leucocytes. Immunocytochemical staining confirmed the IL-1 beta induced ICAM-1 expression on the smooth muscle cells. Moreover, a monoclonal anti-rat ICAM-1 antibody produced a concentration dependent inhibition of the IL-1 beta induced adhesion of monocytes and neutrophils to the smooth muscle cells. CONCLUSIONS: IL-1 beta actively regulates functional ICAM-1 expression in vascular smooth muscle cells. The IL-1 beta-induced expression of ICAM-1 on the smooth muscle cells may be an important contributor to the increased adhesion by monocytes and neutrophils to these cells and suggests that IL-1 beta might play a role in the proinflammatory and immune functions of the modified smooth muscle cells during atherosclerosis and restenosis.

Carvedilol, a new vasodilating beta adrenoceptor blocker antihypertensive drug, protects endothelial cells from damage initiated by xanthine-xanthine oxidase and neutrophils.

OBJECTIVE: Oxygen radical mediated endothelial injury plays an important role in cardiovascular disease. Carvedilol, a new beta blocker and antihypertensive agent, has been shown to have antioxidant activity. The aim of this study was to determine whether carvedilol protects oxygen radical induced endothelial injury. METHODS: Cultured bovine pulmonary artery (BPAEC) and human umbilical vein endothelial cells (HUVEC) were used and oxygen radicals were generated by xanthine-xanthine oxidase or phorbol myristate acetate (PMA) activated human neutrophils. Cell injury was assessed by lactate dehydrogenase (LDH) release and cell death, or 51 Cr release from prelabelled BPAEC. The electron paramagnetic resonance (EPR) spin trapping technique was used to detect the amount of radical spin adducts formed in cell lipids. RESULTS: Carvedilol dose dependently inhibited xanthine-xanthine oxidase induced LDH release from BPAEC and HUVEC, with IC50 values of 3.8 microM and 2.6 microM, respectively, and significantly reduced cell death by xanthine-xanthine oxidase. Other beta blockers tested (propranolol, labetalol, pindolol, and celiprolol) showed a mild effect or no effect at all. Increasing the time of pretreatment with carvedilol enhanced its cell protective effect against oxidative stress. Carvedilol also protected BPAEC dose dependently from PMA activated, neutrophil induced cell injury. Carvedilol had no effect on xanthine oxidase activity. EPR study confirmed that xanthine-xanthine oxidase induced the formation of lipid derived radicals in cell lipids and carvedilol scavenged free radicals, as indicated by the decreased EPR signal. CONCLUSIONS: Carvedilol protects endothelial cells against oxygen radical mediated cell injury and death by scavenging free radicals. The prevention of oxidative injury to endothelial cells might potentially contribute to the clinical beneficial effects of carvedilol as an antihypertensive agent.

Carvedilol, a new antihypertensive agent, prevents lipid peroxidation and oxidative injury to endothelial cells.

The protective effects of carvedilol, a new beta-adrenergic receptor blocker and vasodilating antihypertensive agent, against oxygen free radical-mediated injury were studied in cultured bovine endothelial cells and compared with five other beta-blockers. Carvedilol dose-dependently inhibited oxygen radical-induced lipid peroxidation (50% inhibition at 2.6 mumol/L) and glutathione depletion (50% inhibition at 1.8 mumol/L) in the cells. Under the same conditions, other beta-blockers--propranolol, labetalol, pindolol, atenolol, and celiprolol--had only mild or no effect. Moreover, carvedilol protected against oxygen radical--mediated cell damage, as assessed by cellular lactate dehydrogenase release, with a 50% inhibition at 4.1 mumol/L and increased the cell survival in a dose-dependent manner, whereas other beta-blockers had mild or no effects. Pretreatment of the cells with carvedilol for 7 days significantly enhanced the protective effects of carvedilol. Using 2-methyl-2-nitrosopropane as a trapping agent, the spin adduct in cell lipids was monitored by electron paramagnetic resonance. Carvedilol dose-dependently decreased the intensity of the free radical signals, indicating its free radical-scavenging ability. The prevention of oxidative injury to endothelial cells might potentially contribute to the clinical beneficial effects of carvedilol as an antihypertensive agent.

Expression of interleukin-6, c-fos, and zif268 mRNAs in rat ischemic cortex.

The expression of interleukin-6 (IL-6) mRNA in the focal ischemic rat cortex was studied by means of Northern hybridization. IL-6 mRNA was induced after permanent occlusion of the middle cerebral artery, reached a significant level at 3 h, and peaked at 12 h, i.e., approximately 10-fold increase in the ischemic zone compared with the nonischemic cortex or sham-operated controls. The increased IL-6 mRNA was elevated for at least 24 h. Low levels of IL-6 mRNA were detected in sham-operated rats or in the contralateral nonischemic cortex. The expression of c-fos and zif268 mRNAs, two early response genes, was rapid (increased by 1 h postischemia) and transient (returned to basal levels by 24 and 12 h, respectively), clearly having different kinetic patterns from that of IL-6 mRNA. The early response kinetic pattern of c-fos and zif268 mRNAs in focal ischemia suggests their transcriptional regulatory roles in response to ischemic insult, while the delayed induction pattern of IL-6 mRNA suggests a role for this pleiotropic cytokine in the inflammatory response to the focal ischemic damage of the brain.

Interleukin-1 beta induces expression of adhesion molecules in human vascular smooth muscle cells and enhances adhesion of leukocytes to smooth muscle cells.

Increased expression of cell adhesion molecules is an important pathological event during the development of atherosclerosis. The smooth muscle cell (SMC) is one of the cell types present in the atherosclerotic lesion. To evaluate the regulation of adhesion molecules in human vascular SMCs and its possible role, we studied the expression of adhesion molecules in SMCs stimulated with interleukin 1-beta (IL-1 beta), a pleiotropic cytokine that is involved in the pathological development of vascular diseases including atherosclerosis and restenosis. Our data demonstrated that IL-1 beta markedly induced the adhesiveness of human vascular SMCs for monocytes and neutrophils in a concentration (10 pM - 10 nM)- and time (0.5-24 h)-dependent manner. The maximal induced adhesion by IL-1 beta (1 nM) was reached at 4 h, with 4.6-fold and 3.3-fold for monocytes and neutrophils, respectively. This induction was dose-dependently inhibited by the IL-1 receptor antagonist (IL-1 ra). The IL-1 beta-induced expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin 1 (ELAM-1) on SMCs was examined by reverse transcription/polymerase chain reaction (RT/PCR). Unstimulated, serum-deprived SMCs expressed a low or undetectable level of mRNA for these adhesion molecules. The expression of ICAM-1 and VCAM-1 but not ELAM-1 mRNA was significantly induced with IL-1 beta in a concentration (1 fM - 1 nM)- and time (0.5 - 24 h)-dependent manner. The maximal increase in ICAM-1 and VCAM-1 mRNAs was reached at 4 h after IL-1 beta stimulation. The IL-1 beta-induced adhesion of SMCs for monocytes was partially inhibited by monoclonal anti-human ICAM-1 and anti-human VCAM-1 antibody, but not by anti-human ELAM-1 antibody. Pretreatment of monocytes with anti-human integrin beta 2 antibody significantly reduced the adhesion of monocytes to IL-1 beta-stimulated SMCs. These results suggest that IL-1 beta is a potent inducer for ICAM-1 and VCAM-1 expression in human vascular SMC, and could play a role in the pathogenesis of atherosclerosis by recruitment and retention of inflammatory cells such as monocytes and neutrophils in the lesions.

Carvedilol, a new antihypertensive, prevents oxidation of human low density lipoprotein by macrophages and copper.

Growing evidence indicates that oxidized low-density lipoprotein (LDL) may promote atherogenesis. Therefore, inhibition of LDL oxidation may impede this process. Carvedilol is a vasodilating, beta-adrenoceptor blocking agent. As a new antihypertensive drug, carvedilol is unique by virtue of its potent antioxidant activity. Therefore, we tested the ability of carvedilol to inhibit the oxidation of LDL by either macrophages or Cu2+. Carvedilol inhibited LDL oxidation by macrophages in a dose-dependent manner, with an IC50 value of 3.8 microM, as assessed by a thiobarbituric acid reactive substance (TBARS) assay. Under the same conditions, propranolol showed only a mild inhibitory effect (IC50 > 100 microM), while pindolol, atenolol and labetalol had almost no effect. Carvedilol, at 10 microM, almost completely inhibited the macrophage-induced increase in electrophoretic mobility of LDL, while other beta-blockers at 50-300 microM had no significant effect. Carvedilol inhibited superoxide release from mouse macrophages, which correlated well with its inhibition of LDL oxidation. Carvedilol also inhibited Cu(2+)-induced LDL oxidation with an IC50 value of 17 microM, while all other beta-blockers were inactive up to 300 microM. These observations suggest that carvedilol might not only be an effective antihypertensive drug, but might also be effective in prevention of atherosclerosis.

Characterization of platelet-activating factor-induced elevation of cytosolic free-calcium level in neurohybrid NCB-20 cells.

The effect of platelet-activating factor on the intracellular cytosolic level of free calcium ([Ca2+]i) was studied in neurohybrid NCB-20 cells. In fura-2-loaded NCB-20 cells, platelet-activating factor induced an immediate and concentration-dependent increase in [Ca2+]i with a maximum increase of 334 +/- 27 nM above a basal value of 147 +/- 6 nM (n = 40). Platelet-activating factor-induced [Ca2+]i mobilization was inhibited by the platelet-activating factor antagonists BN 50739, WEB 2086, SRI 63-441 and BN 52021 in a dose-dependent manner with IC50 values of 12, 38, 897 and 45000 nM, respectively. The calcium-channel blockers nifedipine (10 microM) and diltiazem (10 microM) had no effect on the platelet-activating factor-induced increase in [Ca2+]i; however, extracellular Ca(2+)-depletion caused a 63.6 +/- 4.7% reduction of platelet-activating factor-induced increase in [Ca2+]i (n = 5, P less than 0.001). The remaining 36% contributed from intracellular sources was completely inhibited by 10 microM of 8-(N,N-diethylamine)octyl 3,4,5-trimethoxytenzoate hydrochloride (TMB-8). NCB-20 cells exhibited homologous desensitization to sequential addition of platelet-activating factor, but no heterologous desensitization between platelet-activating factor and bradykinin or ATP was observed. These data suggest that activation of the neuronal platelet-activating factor receptor results in an increase in [Ca2+]i primarily via a receptor-operated rather than a voltage-dependent calcium-channel and to a lesser extent from intracellular Ca2+ release. Our findings may contribute to an understanding of the mechanism of platelet-activating factor actions on neuronal cells.

Endothelin receptor binding and cellular signal transduction in neurohybrid NG108-15 cells.

Endothelins are a novel group of potent vasoconstrictor peptides originally isolated from cultured porcine endothelial cells. We and others have previously reported the presence of endothelin receptors in the central nervous system, and this study was designed to further characterize endothelin receptors and their transduction mechanism in cultured neurohybrid NG108-15 cells. Specific binding of [125I]endothelin-1 to NG108-15 cells reached saturation within 60 min at 22 degrees C and was only partially reversible. Scatchard analysis of the saturation binding revealed the presence of one class of high-affinity binding sites with an apparent dissociation constant of 160 pM and a maximal binding capacity of 3.3 x 10(4) sites/cell. Unlabeled endothelin analogues competitively inhibited [125I]endothelin-1 binding to NG108-15 cells and the apparent dissociation constant values obtained from the competition curves correlated well with the EC50 values obtained for inducing elevation of intracellular free Ca2+ level. Endothelin stimulated phosphoinositide metabolism in a dose-dependent manner with an EC50 value of 5.4 nM for inositol trisphosphate formation. The protein kinase C-activator phorbol ester dose-dependently inhibited endothelin-induced phosphoinositide turnover and intracellular free Ca2+ increase, suggesting the involvement of protein kinase C in the regulation of endothelin-induced responses. Neither endothelin-induced phosphoinositide hydrolysis nor endothelin-induced increase in intracellular free Ca2+ were affected by pertussis toxin. These data indicate that endothelin receptors are present on NG108-15 cells and the G protein coupled to endothelin receptor for inducing activation of phospholipase C and increase of free intracellular Ca2+ is insensitive to pertussis toxin.

The influence of maternal protein deficiency on the placental transfer of salicylate in rats.

1 The influence of a low protein diet (5% as compared with a control 21% protein diet) on the placental transfer of sodium salicylate was investigated in Sprague-Dawley rats on day 20 of gestation. 2 Maternal plasma salicylate concentrations (assayed by high pressure liquid chromatography) were generally lower in protein-deficient than in control animals at a wide range of times (0.25 - 12 h) and dose levels (2 - 250 mg/kg, i.v.); however, foetal plasma salicylate levels in the two groups of animals did not differ. 3 The placental transfer of salicylate as indicated by the ratio of foetal plasma or foetal liver to maternal plasma salicylic acid concentration was consistently and significantly greater in the protein-deficient group than in the control group of animals following the administration of the drug to the mother as well as to the foetus. 4 A decrease in calorie without a concomitant decrease in protein intake (pair-fed controls) did not alter the placental transfer of salicylate. 5 The increased placental transfer of salicylate in protein-deficient animals could not be attributed to changes in serum protein-salicylate binding. 6 It is suggested that the pharmacokinetic factors responsible for maintaining a lower level of salicylate in the foetus than in the mother are impaired by maternal malnutrition, and this may increase the foetal effects of maternally ingested salicylate.

Influence of age, sex, pregnancy and protein-calorie malnutrition on the pharmacokinetics of salicylate in rats.

The influence of age, sex, pregnancy and protein-calorie malnutrition (PCM) on the plasma t1/2, plasma clearance (Clp) and apparent volume of distribution (Vd) of sodium salicylate (62 mumol kg-1) was determined in Sprague-Dawley rats. Female and male rats of five different age groups (ages in weeks: pups 1, weanling 3, young 8-9, adult including pregnant 14-15, old 56-60) including three age groups with PCM (8-9, 14-15 and 56-60 weeks old) were used. Plasma and urinary salicylates were assayed by h.p.l.c. Plasma t1/2 was longer and Clp smaller in pups than in weanling and young rats and comparable to values for old rats; Vd of salicylate in pups was larger than in any other group of rats. Plasma t1/2 was longer and Clp as well as Vd of salicylate were smaller in adult females than in males of comparable age. Relative to nonpregnant adult females, Vd of salicylate in pregnant rats was larger but plasma t1/2 and Clp were unchanged. In all groups of rats studied, PCM decreased the plasma t1/2 and increased the Clp of salicylate; Vd was unchanged. Changes in salicylate pharmacokinetics were not due to any differences in serum protein-salicylate binding or to serum testosterone levels. Ovariectomy decreased the plasma t1/2 of salicylate but castration of male rats had no significant effect. Administration of testosterone to ovariectomized female rats exerted no significant effect on salicylate pharmacokinetics. It is concluded that the physiological state and the nutritional status can modify salicylate pharmacokinetics; in so far as the rat model reflects the human situation, these variables should be taken into account for a rational salicylate therapy.

Influence of protein-calorie malnutrition on the pharmacokinetics, placental transfer and tissue localization of dexamethasone in rats.

The influence of protein-calorie malnutrition (PCM) on the pharmacokinetics, transplacental passage and tissue localization of dexamethasone was determined in Sprague-Dawley rats. PCM increased the plasma half-life and volume of distribution of dexamethasone in pregnant but not in nonpregnant rats. Ratios of foetal to maternal serum dexamethasone concentrations were 0.2-0.4 at different dose levels (0.8-20 mumol kg-1), time intervals (0.25-12 h) and gestational ages (day 14-21). PCM increased the foetal serum and tissue concentrations of dexamethasone but exerted no significant effect on its binding to maternal and foetal serum proteins or on its metabolism by the placenta. It is suggested that significantly lower foetal than maternal serum levels of dexamethasone are due to efficient elimination of this agent by the foeto-placental unit and an impairment of this mechanism may account for the observed increase in dexamethasone levels in the foetuses of PCM rats.