Effects of endokinin A/B and endokinin C/D on the antinociception properties of hemopressin in mice

The current study evaluated the effects of hemopressin (HP) on pain modulation by endokinin A/B (EKA/B) and endokinin C/D (EKC/D) at the supraspinal level in mice. Intracerebroventricular administration of HP (10 nmol) fully antagonized the hyperalgesia induced by EKA/B (10, 30, and 100 pmol), and induced a dose-dependent potent analgesic effect. HP at different concentrations (10 pmol, 100 pmol, and 1 nmol) showed varying effects on the analgesic effect of EKA/B (3 nmol). HP extended the duration of the analgesic effect of EKC/D (3 nmol). Moreover, HP at different concentrations (10 pmol, 5 pmol, 1 pmol, and 100 fmol) co-administered with EKC/D (30 pmol) induced significant analgesia at two different time points: 5 min and 50 min. To investigate the antinociceptive mechanism, we used SR140333B and SR142801. HP (1 pmol) potentiated the analgesic effect of SR140333B (100 pmol) + EKA/B (30 pmol) in 5–10 min, while HP (100 pmol) had no effect in the analgesia induced by SR140333B (3 nmol) + EKA/B (3 nmol). HP (1 nmol) fully inhibited the analgesic effect of SR140333B (3 nmol) + EKC/D (3 nmol) or SR142801 (3 nmol) + EKC/D (3 nmol). HP (1 pmol) weakened the analgesic effect of SR142801 (100 pmol) + EKA/B (30 pmol), but HP (100 pmol) strengthened the analgesic effect of SR142801 (3 nmol) + EKA/B (3 nmol). These findings may pave the way for a new strategy on investigating the interaction between tachykinins and opioids on pain modulation.     

Electrophoretic karyotypes of <Emphasis Type="Italic">C. neoformans</Emphasis> serotype A recovered from Thai Patients with AIDS

Thirty-seven clinical isolates of C. neoformans were recovered from AIDS patients and all were serotype A according to standard typing tests. They were further analyzed using RAPD, PCR fingerprinting, and PFGE along with 2 additional reference isolates ATCC 34871 (serotype A) and RV 45981 (serotype D). Using 2 different RAPD primers, all of the clinical isolates and the reference serotype A (ATCC 34871) gave similar RAPD patterns while serotype D (RV 45981) gave distinctive pattern. Corresponding result was also obtained upon PCR by using a primer for microsatellite (GACA)₄. However, using a primer specific to minisatellite M13 + 1, all PCR fingerprinting gave similar gel patterns (M1) for 35/37 of the clinical isolates and the reference serotype A while two clinical isolates generated different patterns called M2 and M3. The reference serotype D gave distinctive pattern called M4. PFGE gave 17 different karyotypes that could be categorized into 4 groups named EKA (1–6), EKB (1–5), EKC (1– 5) and EKD (1). The reference serotype A fell into group EKA as EKA6 while the reference serotype D fell into group EKC as EKC5. Among the clinical isolates, EKA group (20/37 isolates) and type EKA1 (16/20) dominated with only one isolate each for types EKA2 to EKA5. The next most prevalent was group EKB (12/37 isolates) which dominately fell in type EKB1 (8/12) and only one isolate each for types EKB2 to EKB5. Group EKC (4/37 isolates) and group EKD (1/37) had only one isolate for each type (EKC1 to EKC 4 and EKD1). The 2 predominant karyotypes (EKA1, 16/37 and EKB1, 8/37) may represent two originally common clones of C. neoformans expose among the patients. The high discriminatory power of PFGE infers the benefit of subtyping which lead to better understanding on the epidemiology and pathogenic potential of C. neoformans subtypes. Moreover, PCR fingerprinting and RAPD infer the feasibility of detail analysis between serotypes A and D for unencapsulated C. neoformans.     

Pharmacological characteristics of endokinin C/D-derived peptides in nociceptive and inflammatory processing in rats

Endokinins designated from the human TAC4 gene consist of endokinin A, endokinin B, endokinin C (EKC) and endokinin D (EKD). EKC/D is a peptide using the common carboxyl-terminal in EKC and EKD and consists of 12 amino acids, and exerts antagonistic effects on the induction of scratching behavior by substance P (SP). Some of SP-preferring receptor antagonists have several d-tryptophan (d-Trp); however, the pharmacological effect of EKC/D-derived peptides with d-Trp remains to be solved. Therefore, to clarify the pharmacological characteristics of EKC/D-derived peptides, effects of pretreatment with these peptides on SP-induced scratching and thermal hyperalgesia, formalin-induced flinching and carrageenan-induced inflammation were evaluated. Intrathecal administration of [d-Trp(8)]-EKC/D and [d-Trp(10)]-EKC/D showed a markedly long inhibitory effect, at least 14 h, whereas the antagonistic effects of [d-Trp(8,10)]-EKC/D and EKC/D without d-Trp disappeared after 1h. Furthermore, the inhibitory effect of [d-Trp(10)]-EKC/D-derived peptides was dependent on the number of amino acids from the amino-terminus, and the more numerous the amino acids, the more marked the antagonistic effect. Thus, these results indicate that the effective duration of EKC/D-derived peptides is dependent on the number of d-Trp in the carboxyl-terminal region and the amino-terminal region regulates the antagonistic effect of EKC/D.     

Effect of the carboxyl-terminal of endokinins on SP-induced pain-related behavior

The preprotachykinin C gene encodes four endokinins, A, B, C, and D. Endokinins A and B and substance P (SP) are typical tachykinin peptides since their carboxyl-terminal regions share an F-F-G-L-M-amide, while endokinins C and D share an F-Q-G-L-L-amide. It is demonstrated that pretreatment with a peptide consisting of a common sequence between endokinins C and D (EKC/D) attenuates the induction of scratching behavior and thermal hyperalgesia by intrathecal administration of SP or EKA/B (the carboxyl-terminal dacapeptide common in endokinins A and B), suggesting that leucine at the carboxyl-terminal of EKC/D may have a crucial role in eliciting these effects. When the effect of [Leu¹¹]-SP and [Leu¹⁰]-EKA/B on SP-induced pain-related behavior was examined, the induction of pain-related behavior was markedly attenuated by pretreatment with these peptides. This indicates that leucine at the carboxyl-terminal of these peptides plays a crucial role in eliciting this antagonistic effect.     

Involvement of the nitric oxide/L-arginine and sympathetic nervous systems on the vasodepressor action of human urotensin II in anesthetized rats

This study examined if the nitric oxide (NO)/L-arginine pathway participates in and if the sympathetic nervous system attenuates the depressor action of human urotensin II. I.V. bolus injections of human urotensin II (0.1-30 nmol/kg) caused dose-dependent decreases in mean arterial pressure (MAP, EC(50) = 2.09 +/- 0.8 nmol/kg; Emax = -18 +/- 3 mmHg ) and increases in heart rate. The depressor response to human urotensin II (3 nmol/kg) was attenuated by approximately 50% in rats with MAP elevated through pretreatment with N(G)-nitro-L-arginine methyl ester (inhibitor of NO synthase), relative to that in rats with MAP elevated to a similar level through a continuous infusion of noradrenaline. Autonomic blockade with i.v. injections of mecamylamine (ganglion blocker) and propranolol (beta-adrenoceptor antagonist) markedly augmented the depressor response to human urotensin II, but almost completely attenuated the tachycardia. The results suggest that the depressor response to human urotensin II is partially mediated via the NO/L-arginine pathway, and is suppressed by activity of the sympathetic nervous system. Furthermore, tachycardic response to human urotensin II is primarily mediated indirectly via baroreflex mechanisms.     

苍术素对大鼠心脏正性肌力的作用及其机制

目的:研究中药苍术的有效成分苍术素(Atractylodin)的正性肌力作用及其机理。方法:随机选取6只雄性SD大鼠进行在体压力-容积环(P-V loop)实验,加药前为Control组,经腹腔注射苍术素(3 mg/kg)后为苍术素组(自身对照),分析6只雄性大鼠加药后对大鼠左心室心输出量、容积及动脉压的作用;大鼠离体心脏灌流实验中,依次灌流给药:第一部分为Control→0.1→1→10μmol/L苍术素浓度梯度灌流,第二部分为Control→200 nmol/L H89 (PKA抑制剂)→200 nmol/L H89+10μmol/L苍术素,第三部分为Control→500 nmol/L KN-93 (Ca MKII抑制剂)→500 nmol/L KN-93+10μmol/L苍术素,第四部分为Control→10 nmol/L Calyculin A (PP1,PP2A抑制剂)→10nmol/L Calyculin A+10μmol/L苍术素,加药前的正常空白组为Control组,分析每部分各6只雄性大鼠的组间左心室发展压的变化;在大鼠心肌细胞钙释放实验中,分组、给药的方法和浓度同...     

SR 59230A对心衰大鼠心脏MicroRNAs表达的影响

目的：观察β3肾上腺素受体（β3-AR）对心衰大鼠心脏MicroRNAs表达的影响及可能的作用机制。方法：大鼠冠脉左前降支结扎造成心衰模型,假手术大鼠只穿线不结扎。造模成功大鼠再随机分为：心衰组（CHF control group）和心衰+SR 59230A组（CHF+SR group）;假手术大鼠也随机分为假手术组（Sham group）和假手术+SR 59230A组（Sham+SR group）。Sham+SR组和CHF+SR组每天两次腹腔注射SR （85 mmol/L,1 ml）,连续注射7周。结果：①miScript miRNA PCR Arrays显示,在体阻断β3-AR后,假手术组与心衰组有18种MicroRNAs共同表达下调;经文献比对,与NF-κB相关的MicroRNAs有6种,分别为miR-125b-5p,miR-143-3p,miR-145-5p,miR-26a-5p,miR-30a-5p和miR-320-5p。②大鼠心脏组织切片观察到NF-κB在心衰大鼠心肌细胞核与细胞质中均有分布,而p53在心肌细胞质分布较多,NF-κB和p53表达明显高于假手术组（P<0.05）。阻断β3-AR后,心衰组心脏NF-κB和p53表达显著减少（P<0.05）,而假手术组NF-κB和p53表达略增加（P<0.05）。③Western blot结果发现心衰大鼠NF-κB p65表达高于假手术组（P<0.05）,给予β3-AR阻断剂后,心衰组心脏NF-κB p65和p53-Phospho-Serine 15表达均下降（P<0.05）,而假手术组心脏阻断β3-AR后,NF-κB、p53和p53-Phospho-Serine 15表达均增加（P<0.05）。结论：阻断β3肾上腺素受体有利于缓解心衰大鼠心脏的损伤;β3-AR可引起MicroRNAs表达变化且与NF-κB信号通路有关。     

Role of Serotonin2A and Serotonin2B/2C Receptor Subtypes in the Control of Accumbal and Striatal Dopamine Release Elicited In Vivo by Dorsal Raphe Nucleus Electrical Stimulation

This study investigates, using in vivo microdialysis, the role of serotonin2A (5-HT2A) and 5-HT(2B/2C) receptors in the effect of dorsal raphe nucleus (DRN) electrical stimulation on dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) extracellular levels monitored in the nucleus accumbens (NAC) and the striatum of halothane-anesthetized rats. Following DRN stimulation (300 microA, 1 ms, 20 Hz, 15 min) DA release was enhanced in the NAC and reduced in the striatum. The 5-HT2A antagonist SR 46349B (0.5 mg/kg) and the mixed 5-HT(2A/2B/2C) antagonist ritanserin (0.63 mg/kg) significantly reduced the effect of DRN stimulation on DA release in the NAC but not in the striatum. DA responses to DRN stimulation were not affected by the 5-HT(2B/2C) antagonist SB 206553 (5 mg/kg) in either region. None of these compounds was able to modify the enhancement of DOPAC and 5-HIAA outflow induced by DRN stimulation in either the NAC or the striatum. Finally, in both brain regions basal DA release was significantly increased only by SB 206553. These results indicate that 5-HT2A but not 5-HT(2B/2C) receptors participate in the facilitatory control exerted by endogenous 5-HT on accumbal DA release. Conversely, 5-HT(2B/2C) receptors tonically inhibit basal DA release in both brain regions.     

Nphe1]nociceptin(1-13)-NH2 antagonizes nociceptin-induced hypotension, bradycardia, and hindquarters vasodilation in the anesthetized rat

The purpose of this study was to investigate the effects of [Nphe1]nociceptin(1-13)-NH2 on nociceptin-induced decreases in mean arterial pressure (MAP), heart rate (HR), and hindquarters vascular bed resistance (HVBR) of the anesthetized rat. The results showed that i.c.v. or i.v. [Nphe1]nociceptin(1-13)-NH2 (1.5-12 nmol/kg and 5-120 nmol/kg, respectively) could antagonize the depressor effects of i.c.v. or i.v. nociceptin (3 and 30 nmol/kg, respectively) on MAP and HR. Furthermore, [Nphe1]nociceptin(1-13)-NH2 (5-120 nmol/kg) could reverse nociceptin (30 nmol/kg)-induced decrease of HVBR. However, [Nphe1]nociceptin(1-13)-NH2 had no significant effects on similar effects induced by morphine. Our results suggest that [Nphe1]nociceptin(1-13)-NH2 acts as a selective antagonist of the nociceptin receptor in the cardiovascular system of the rat.     

Lipopolysaccharide directly stimulates aldosterone production via toll‐like receptor 2 and toll‐like receptor 4 related PI3K/Akt pathway in rat adrenal zona glomerulosa cells

The level of circulating endotoxin is related to the severity of cardiovascular disease. One of the indexes for the prognosis of cardiovascular disease is the plasma aldosterone level. Recently, the Toll‐like receptors (TLRs), lipopolysaccharide (LPS)‐regulated receptors, were found not only to mediate the inflammatory response but also to be important in the adrenal stress response. Whether LPS via TLRs induced aldosterone production in adrenal zona glomerulosa (ZG) cells was not clear. Our results suggest that LPS‐induced aldosterone secretion in a time‐ and dose‐dependent manner and via TLR2 and TLR4 signaling pathway. Administration of LPS can enhance steroidogenesis enzyme expression such as scavenger receptor‐B1 (SR‐B1), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage (P450scc) enzyme. LPS‐induced SR‐B1 and StAR protein expression are abolished by TLR2 blocker. Furthermore, we demonstrated that phosphorylation of Akt was elevated by LPS treatment and reduced by TLR2 blockers, TLR4 blockers, and LY294002 (PI₃K inhibitor). Those inhibitors of PI₃K/Akt pathways also abolish LPS‐induced aldosterone secretion and SR‐B1 protein level. In conclusion, LPS‐induced aldosterone production and SR‐B1 proteins expression are through the TLR2 and TLR4 related PI₃K/Akt pathways in adrenal ZG cells. J. Cell. Biochem. 111: 872–880, 2010. © 2010 Wiley‐Liss, Inc.     

Comparative effect of Phoneutria nigriventer spider venom and capsaicin on the rat paw oedema

Capsaicin, the pungent component of hot peppers, and the venom of the spider Phoneutria nigriventer are able to activate sensory nerves resulting in cutaneous neurogenic plasma extravasation. This study was undertaken to compare the ability of these substances to evoke oedema in the rat hind-paw and mechanisms underlying this effect. Subplantar injection of either Phoneutria nigriventer venom (PNV; 1-100 microg/paw) or capsaicin (10-200 microg/paw) caused a significant paw oedema that was potentiated by CGRP (10 pmol/paw). In rats treated neonatally with capsaicin to deplete neuropeptides, the paw oedema induced by either PNV (100 microg/paw) or capsaicin (100 microg/paw) was partially reduced (P<0.05). The tachykinin NK1 receptor antagonist SR140333 (0.2 micromol/kg; i.v.) prevented the paw oedema induced by the tachykinin NK1 receptor agonist GR73632 (30 pmol/paw) and partially reduced paw oedema induced by PNV or capsaicin. Treatment of rats with compound 48/80 (5 mg/kg; s.c. 3 days) or with both H1 receptor antagonist (mepyramine; 1 nmol/paw) and 5-HT receptor antagonist (methysergide; 1 nmol/paw) significantly inhibited PNV- or capsaicin-induced paw oedema. The combined treatment with mepyramine and methysergide and SR140333 further reduced PNV- and capsaicin-induced paw oedema. The bradykinin B2 receptor antagonist Hoe 140 affected neither PNV- nor capsaicin-induced responses. Our results suggest that PNV and capsaicin each induce paw oedema that is partially mediated by activation of sensory fibers culminating in the release of substance P as well as by activation of mast cells which in turn release amines such as histamine and 5-HT.     

Elevated P/Q type (alpha1A) and L2 type (alpha1D) Purkinje cell voltage-gated calcium channels in the cerebella of seizure prone gerbils

Differences in expression of N-methyl-D-aspartate (NMDA) receptor and voltage gated Ca2+ channels (VGCC) in the gerbil cerebellum were investigated to identify routes of Ca2+ influx that may be involved in Purkinje cell damage. Immunodensities of NR1 and NR2A/B were the same in seizure resistant (SR) and seizure sensitive (SS) gerbils. However, both P/Q type (alpha1A) and L2 type (alpha1D) VGCC levels were higher in the Purkinje cells of SS gerbils than in those of SR gerbils, whereas N type (alpha1B) and L1 type (alpha1C) VGCC levels were similar in the two groups. Our findings suggest that increases in P/Q type (alpha1A) and L2 type (alpha1D) VGCC are implicated in the degeneration of Purkinje cells in SS gerbils.     

Cardiac and vascular actions of sarafotoxin S6b and endothelin-1

Snake venom-derived sarafotoxin S6B (SRT) and porcine endothelium-derived endothelin-1 (ET) have striking structural similarities. In conscious, freely-moving rats, ET (0.67 nmol/kg) produced a transient tachycardia and fall in arterial blood pressure which was followed by a long-lasting increase in arterial pressure, bradycardia, decrease in cardiac output (CO) and marked increase in total peripheral resistance. In contrast, SRT (0.67 nmol/kg) produced only the sustained cardiovascular responses. The sustained cardiovascular effects of SRT or ET were similarly attenuated by nifedipine. SRT and ET (30 nM) produced vasoconstriction in the isolated perfused mesenteric vascular bed without initial vasodilation. SRT and ET had potent positive inotropic and negative chronotropic effects on isolated perfused hearts and induced toxic reactions including coronary vasospasm, arrhythmias, A-V block and ventricular fibrillation. In addition to SRT lacking the initial depressor response in vivo, several differences in the activities of the peptides were also observed. ET produced greater and longer-lasting actions than SRT in producing pressor and vasoconstrictor responses in all 3 preparations, and in its ability to induce toxic effects on the heart.     

Attenuated hypotensive response to proadrenomedullin N-terminal 20 peptide in pregnant rats: modulation by steroid hormones

The hypotensive effect of proadrenomedullin N-terminal 20 peptide (PAMP) was examined in conscious pregnant (8, 14, and 20 days of pregnancy) and nonpregnant rats. Intravenous administration of PAMP (3–60 nmol/kg) produced a dose-dependent depressor response in both pregnant and nonpregnant rats. However, the maximum decrease in blood pressure was significantly attenuated in pregnant rats in mid- and late-gestation (14 and 20 days), but not in early gestation (8 days), than in nonpregnant rats. In ovariectomized rats, the depressor responses in 17β-estradiol (E2)-treated, progesterone (P)-treated, and E2+P-treated rats were significantly attenuated compared with the control rats. We also demonstrated that treatment of sex hormones reduces the depressor response to PAMP in 8-day pregnant rats. In addition, we showed that treatment of sex hormone receptor antagonists partially prevents the attenuation of the depressor response to PAMP in 20 day pregnant rats. These findings suggested that the hypotensive response to PAMP was more attenuated in pregnant rats in mid- and late-gestation than in nonpregnant rats, and that the changes in depressor response that occur at term in pregnant rats may be mediated by sex hormones. PAMP may play some important role in cardiovascular regulation during pregnancy.     

Ca2+ displacement by polymyxin B from sarcolemma isolated by ‘gas dissection’ from cultured neonatal rat myocardial cells

Amphiphilic, cationic Polymyxin B is shown to displace Ca²⁺ from ‘gas dissected’ cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca²⁺ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca²⁺, respectively. Total Ca²⁺ displaced by a non-specific cationic probe, lanthanum (La³⁺), at maximal displacing concentration (1 mM) was $\text{0.172 ± 0.02}\text{nmol/μg}$ membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La³⁺-displaceable Ca²⁺ or $\text{0.072 ± 0.01}\text{nmol/μg}$ protein. 5 mM Polymyxin displaced Ca²⁺ in amounts equal to those displaced by 1 mM La³⁺. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La³⁺-displaceable Ca²⁺ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La³⁺-displaceable Ca²⁺ to $\text{0.227 ± 0.02}\text{nmol/μg}$ protein ($\text{P < 0.05}$), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca²⁺ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca²⁺-binding sites in natural membranes.     

Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing

SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS.     

Blockade of cardiac sarcoplasmic reticulum K+ channel by Ca2+: two-binding-site model of blockade.

Potassium countercurrent through the SR K+ channel plays an important role in Ca2+ release from the SR. To see if Ca2+ regulates the channel, we incorporated canine cardiac SR K+ channel into lipid bilayers. Calcium ions present in either the SR lumenal (trans) or cytoplasmic (cis) side blocked the cardiac SR K+ channel in a voltage-dependent manner. When Ca2+ was present on both sides, however, the block appeared to be voltage independent. A two-binding site model of blockade by an impermeant divalent cation (Ca2+) can explain this apparent contradiction. Estimates of SR Ca2+ concentration suggest that under physiological conditions the cardiac SR K+ channel is partially blocked by Ca2+ ions present in the lumen of the SR. The reduction in lumenal [Ca2+] during Ca2+ release could increase K+ conductance.     

Synthesis and structure-activity relationships of analogs of EM-652 (acolbifene), a pure selective estrogen receptor modulator. Study of nitrogen substitution

EM-652 (acolbifene) analogs have been synthesized as selective estrogen receptor modulators. Substitution on the nitrogen atom of these 2H-1-benzopyran derivatives has been studied for its influence on antiestrogenic activity. Binding to the rat estrogen receptor, inhibition of estradiol-stimulated proliferation of T-47D breast cancer cells, as well as antiuterotrophic and uterotrophic activities in ovariectomized mice have been evaluated. 2H-1-Benzopyran 1b (EM-343, racemic form of EM-652), which contains a piperidine ring, shows the best pharmacological profile; RBA = 380, IC50 value = 0.110 nM (in T-47D cells), as well as 63% and 84% antiuterotrophic inhibitions at the 7.5 and 75 nmol doses, respectively.     

Ca2+ displacement by Polymyxin B from sarcolemma isolated by 'gas dissection' from cultured neonatal rat myocardial cells

Amphiphilic, cationic Polymyxin B is shown to displace Ca2+ from 'gas dissected' cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca2+ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca2+, respectively. Total Ca2+ displaced by a non-specific cationic probe, lanthanum (La3+), at maximal displacing concentration (1 mM) was 0.172 +/- 0.02 nmol/microgram membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La3+-displaceable Ca2+ or 0.072 +/- 0.01 nmol/microgram protein. 5 mM Polymyxin displaced Ca2+ in amounts equal to those displaced by 1 mM La3+. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La3+-displaceable Ca2+ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La3+-displaceable Ca2+ to 0.227 +/- 0.02 nmol/microgram protein (P less than 0.05), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca2+ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca2+-binding sites in natural membranes.