Desensitization by protein kinase C activation differentially uncouples formyl peptide receptors from effector enzymes in HL-60 granulocytes

The hypothesis that protein kinase C (PKC) participates in agonist-mediated desensitization of formyl peptide receptors in HL-60 granulocytes was tested. fMet-Leu-Phe and leukotriene B₄(LTB₄) produced homologous desensitization of agonist-stimulated intracellular calcium transients. Pre-treatment with the PKC activator, phorbol myristate acetate (PMA; 10 nM), abolished both fMet-Leu-Phe and LTB₄-stimulated calcium transients. Membranes prepared from control HL-60 granulocytes (NM) or cells treated with 10 nM PMA (PMA-M) demonstrated increased formyl peptide receptor and G protein density, as determined by radioligand binding and pertussis toxin- and cholera toxin-catalysed ADP ribosylation. fMet-Leu-Phe stimulation of guanosine 5′-[γ-thio]-triphosphate (GTPγS) binding and GTP hydrolysis and GDP inhibition of fMet-Leu-Phe binding were not different between NM and PMA-M. Pre-treatment with 10 nM PMA did not inhibit subsequent fMet-Leu-Phe-stimulated superoxide generation or phospholidase D activation. We conclude that PKC desensitizes fMet-Leu-Phe-stimulated phospholipase C, but not phospholipase D, responses and that PKC activation does not mediate agonist-induced desensitization of formyl peptide receptors.     

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca²⁺ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC₅₀) and maximal inhibition of BK induced an increase in [Ca²⁺]_i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca²⁺ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K_D and B_max of the BK receptor for binding (control: K_D = 1.7 ± 0.2 nM; B_max = 47.3 ± 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca²⁺ mobilisation in VSMCs.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Effect of protein kinase C activation on agonist-mediated phosphositide metabolism in rabbit retinal cells

4β-Phorbol 12-myrisate 13-acetate (PMA), a tumour-promoting phorbol ester, and 1-oleoly-2-acetylglycerol (OAG), a synthetic diacylglycerol, induced an inhibition of muscarinic and ₁-adrenergic receptor-mediated stimulation of PIP₂ breakdown and IPs accumulation in both rabbit retinal slices and primary retinal cultures. Furthermore, an increase in [Ca²⁺]_i, mediated by activation of these receptors in 3–5 and 25–30 day old rabbit retinal cultures, was also inhibited by PMA. Neither PMA nor OAG had an effect on the serotonin-mediated PIP₂ breakdown, IPs accumulation or Ca²⁺ mobilization. Although A23187 also stimulated IPs formation by acting directly on phospholipase C, PMA had no effect. Maximal inhibition of the carbachol- and noradrenaline-mediated responses was achieved with a 15 min preincubation with PMA at concentrations of 0.1 and 0.01 μM in retinal slices and primary retinal cultures, respectively. Neither PMA nor OAG influenced the basal levels of phosphoinositides, IPs or [Ca²]_i. In addition, the inactive phorbol ester, 4-phorbol 12,13-didecanoate, had no effect on any of the agonist-induced responses. Staurosporine, a potent inhibitor of protein kinase C, significantly attenuated the inhibitory effects exerted by PMA and OAG. These results suggest that calcium- and phospholipid-dependent protein kinase, which is activated by either PMA or OAG, exert inhibitory effects on muscarinic and ₁-adrenergic responses. This modulatory feedback “down regulation” role by PKC does not, however, affect serotonergic mediated responses, and thus exhibits a certain selectivity about the site of action. The possible mechanism(s) by which PKC induces its actions are discussed.     

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.     

Altered role of C-type natriuretic peptide-activated pGC-cGMP-PDE3-cAMP signaling in hyperthyroid beating rabbit atria

The role of C-type natriuretic peptide (CNP) in the pathophysiology of atrial function in hyperthyroidism has not been defined. This study was to define the role of CNP-activated particulate (p) guanylyl cyclase (GC)-cGMP-phosphodiesterase (PDE)3 signaling in the regulation of cAMP levels and contractile and secretory functions in the atria from hyperthyroid rabbits. Experiments were performed in perfused beating rabbit atria. CNP was used to activate pGC. In euthyroid atria from sham-treated rabbits, CNP (100 nM) increased cGMP and cAMP efflux by 176.7+/-17.7 and 55.3+/-10.0%, respectively. CNP decreased stroke volume and pulse pressure and ANP release by 51+/-7 and 41+/-2 and 60.4+/-3.2%, respectively. Pretreatment with milrinone blocked the CNP-induced increase of cAMP but without significant changes in decrease of atrial dynamics and ANP release. In hyperthyroid atria, CNP-induced increase of cGMP levels was accentuated, while CNP-induced increase of cAMP was attenuated. The gain of cAMP, i.e., change in cAMP efflux concentration in terms of cGMP was attenuated in the hyperthyroid compared to euthyroid atria. CNP rather increased atrial dynamics in hyperthyroid atria instead of decrease. CNP-induced decrease in atrial ANP release was attenuated. Pretreatment with milrinone blocked the CNP-induced increase of cAMP levels concomitantly with a decrease of atrial dynamics. The present study demonstrates that altered role of CNP-activated pGC-cGMP-PDE3-cAMP signaling is involved in the pathophysiology of hyperthyroid heart.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

C-type natriuretic peptide induces human colonic myofibroblast relaxation

Intestinal response to injury requires coordinated regulation of the tension exerted by subepithelial myofibroblasts (SEM). However, the signals governing relaxation of intestinal SEM have not been investigated. Our aim was to test the hypothesis that signal transduction pathways initiated by C-type natriuretic peptide (CNP) induce intestinal SEM relaxation. We directly quantified the effects of CNP on isometric tension exerted by cultured human colonic SEM. We also measured the effects of CNP on cGMP content, myosin regulatory light chain (MLC) phosphorylation, and cytosolic Ca2+ concentration. CNP induced relaxation of SEM within 10 s. By 10 min, relaxation reached a plateau that was sustained for 2 h. CNP-induced relaxation was saturable, with a maximal decrease in tension (51.7 +/- 3.8 dyn) observed at 250 nM. SEM relaxation in response to CNP constituted approximately 23% of total basal tension. CNP increased intracellular cGMP content and reduced MLC phosphorylation. Effects of CNP on cGMP and MLC exhibited the same dose dependence as CNP-induced relaxation. MLC phosphorylation decreased within 2 min of CNP exposure and was sustained for at least 45 min. CNP also stimulated a large transient increase in cytosolic Ca2+ concentration that occurred within 30 s and was nearly complete by 1 min. We also observed that calyculin-A, a potent inhibitor of MLC phosphatase, completely abolished the reduction in MLC phosphorylation induced by CNP. These results suggest that CNP induces intestinal SEM relaxation through cGMP-associated reductions in MLC phosphorylation. Moreover, these findings raise the possibility that CNP plays a role in intestinal wound healing.     

Endothelin Stimulates Phospholipase D in Striatal Astrocytes

Abstract: In primary cultures of mouse striatal astrocytes prelabeled with [³H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [³H]phosphatidic acid and [³H]diacylglycerol. In the presence of ethanol, a production of [³H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC₅₀ = 2–5 n M ). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a G_i/G_o protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [³H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally depended on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-hydroxy-1-methoxy-Delta(8)-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

Synthesis and opioid activity of N,N-dimethyl-Dmt-Tic-NH-CH(R)-R' analogues: acquisition of potent delta antagonism

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (K_i=0.035–0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′–8′) elicited a greater reduction in μ-affinity (40–70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′–7′) (pA₂=8.14–9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA₂=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.     

Synthesis of non-immunosuppressive cyclophilin-Binding cyclosporin A derivatives as potential anti-HIV-1 drugs

Original cyclosporin A (CsA) derivatives bearing various alkylthio side chains at the sarcosine residue 3 (R configuration) and for the most potent and selective compounds a 4′-hydroxyl group at the Me-Leucine residue 4 were prepared in one or two steps from commercially available CsA. The [2-(dimethyl or diethylamino)-ethylthio-Sar]³-[(4′-OH)MeLeu]⁴-CsA derivatives 3k and 3l displayed potent in vitro anti-HIV-1 (IC₅₀ 46 nM) and low immunosuppressive activities (IC₅₀≥1500 nM).     

Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae)

A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N′-propylurea and N-hexadecyl-N′-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC₅₀ around 90 nM. The importance of substitution number and type was investigated and results indicated that N, N′-disubstitution with asymmetric alkyl groups was favored. Potencies of pharmacophores decreased as follows: amide>urea>carbamate>carbodiimide>thiourea and thiocarbamate for N, N′-disubstituted compounds with symmetric substituents, and urea>amide>carbamate for compounds with asymmetric N, N′-substituents. JHEH hydrolyzes t-DPPO with a K_m of 65.6 μM and a V_max of 59 nmol min⁻¹ mg⁻¹ and has a substantially lower K_m of 3.6 μM and higher V_max of 322 nmol min⁻¹ mg⁻¹ for JH III. Although none of these compounds were potent inhibitors of hydrolysis of JH III by JHEH, they are the first leads toward inhibitors of JHEH that are not potentially subject to metabolism through epoxide degradation.     

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (, β₁, β₂, γ, δ and ) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10 mM NaN₃, combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β₁, δ and isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by , β₁, γ, δ and were maintained and even potentiated, moreover, an increase in β₂ (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC isoform, which following reperfusion was found only in the cytosol. PKC β₁ and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β₂ and isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 1 mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β₁ isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β₂ and isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.     

Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts

Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC₅₀) and maximal, saturable binding (B_max) were estimated from Scatchard analyses and were found to be 24.2 ± 3.3 pM and 56500 ± 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of 100 nM to a peak level of 600–800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC₅₀ of 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of ⁴⁵Ca from cells loaded to isotopic equilibrium (3 h) with ⁴⁵Ca. The intracellular second messenger, IP₃, also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with ⁴⁵Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 ± 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP₃-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.     

Prooxidant activity of β-carotene under 100% oxygen pressure in rat liver microsomes

The effects of the partial pressure of oxygen (pO₂ on antioxidant efficiency of β-carotene in inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced lipid peroxidation are investigated in rat liver microsomal membranes. The rate of peroxyl radicals generated by thermolysis of AAPH at 37°C is markedly higher at 150 than 760 mm Hg pO₂. At 150 mm Hg pO₂ β-carotene acts as an antioxidant, inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced Malondialdehyde (MDA) formation. At 760 mm Hg pO₂, it loses its antioxidant activity and shows a prooxidant effect, increasing lipid peroxidation products, -Tocopherol prevents the prooxidant effect of β-carotene in a dose-dependent manner. Our data provide the first evidence of a prooxidant effect of β-carotene under 100% oxygen pressure in a biological membrane model and point out the existence of cooperative interactions between β-carotene and -tocopherol.     

Unsymmetric nonpeptidic HIV protease inhibitors containing anthranilamide as a P2′ ligand

A series of novel unsymmetrical anthranilamide-containing HIV protease inhibitors was designed. The structure-activity studies revealed a series of potent P2–P3′ inhibitors that incorporate an anthranilamide group at the P2′ position. A reduction in molecular weight and lipophilicity is achieved by a judicious choice of P2 ligands (i.e., aromatic, heteroaromatic, carbamate, and peptidic). A systematic investigation led to the 5-thiazolyl carbamate analog 8m, which exhibited a favorable C_max/EC₅₀ ratio (>30), plasma half-life (>8 h), and potent in vitro antiviral activity (EC₅₀ = 0.2 uM).     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.