Localization of α1-adrenoceptors in rat and human hearts by immunocytochemistry

The localization of the ai adrenoceptors (₁-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human ₁-AR (AS sequence 192–218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, ₁-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of ₁-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the -adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.     

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups

The triated adrenergic antagonists Prazosin ([³H]PRZ) and Idazoxan ([³H]IDA, or RX-781094) bind specifically and with high affinity to ₁ and ₂-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (K _d) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (–SS–) bridges and sulfhydryl (–SH) groups in the specific combination of [³H]PRZ and [³H]IDA to the ₁ and ₂-adrenoceptors. Pretreatment of the membranes with the –SS– reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [³H]IDA binding (₂-sites) was more sensitive to both DTT and NEM than the [³H]PRZ sites (₂-adrenoceptors), and the initial changes induced by alkylation of the ₂-site were due to an important decrease in the affinity for [³H]IDA, as judged by the increase in theK _d. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the ₂-site. The results provide evidence for the participation of –SS– and –SH groups in the binding site of ₁ and ₂-adrenoceptors in the cerebral cortex.     

Lymphosarcoma-induced alterations in hepatic adrenergic receptors: Implications to the hypoglycemia of cancer cachexia

A highly malignant transplantable rat lymphosarcoma was studied to determine the involvement of hepatic adrenergic receptors in the development of the hypoglycemia of cancer cachexia. Following inoculation of Fischer 344 rats with lymphosarcoma cells, rats were examined at 2 and 4 weeks, at the pre-cachexic stage; 6 weeks, at the transitional stage; and 7 weeks, at the cachexic hypoglycemic stage of lymphosarcoma progression. Death occurred by the 8th week. Blood glucose levels in lymphosarcoma-bearing rats relative to control rats were: unaffected at week 2; significantly reduced 8% at weeks 4 and 6; and reduced 24% at week 7. ₁ adrenergic receptor binding to plasma membranes isolated from the livers of lymphosarcoma-bearing rats was: 114, 89, 67 and 30% of control at weeks 2, 4, 6, and 7, respectively. Kinetic analysis indicated that the lymphosarcoma-induced decrease at week 7 was due to a decrease in numbers of receptors with no change in affinity: Bmax_control: 1411.1 fmol/mg; Kd_control: 0.44 nm; Bmax_lympho: 345.5 fmol/mg; Kd_lympho: 0.50 nm. ₂ adrenergic receptor binding to plasma membranes isolated from the livers of lymphosarcoma-bearing rats was: 130, 137, 243 and 212% of control at weeks 2, 4, 6, and 7, respectively. The pattern of changes in hepatic ₁, ₂ and adrenergic receptors at week 6 was comparable to that of 17 day fetal liver: a decrease in ₁ and and an increase in ₂. Hepatic adrenergic receptor changes occurred in the absence of liver damage and were not due to contamination of the liver plasma membrane fractions with lymphosarcoma cells. Plasma insulin levels displayed modest (10–15%), but not statistically significant, increases post-inoculation after week 4. Plasma glucagon levels fluctuated post-inoculation until week 7 where they were significantly increased: 202% of control. Plasma T₃ and T₄ levels displayed an early and steady decline after lymphosarcoma inoculation: T₃: unchanged at week 2 and significantly decreased 14, 44 and 50% at weeks 4, 6 and 7, respectively. T₄ increased 20% at week 1; decreased 9% at week 4 and significantly decreased thereafter: 55 and 49% at weeks 6 and 7, respectively. We propose that the development of the hypoglycemia of cancer cachexia in this lymphosarcoma model is due primarily to an early and progressive thyroid hormone dependent decrease in the number of hepatic ₁ adrenergic receptors, compounded by an increase and decrease, respectively, in the hepatic and ₂ adrenergic receptors.     

Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

The effects of interleukin-1 therapy on peripheral blood granulocyte function in humans

During a phase I trial of interleukin-1 (IL-1) in patients with ovarian carcinomas, the effects of this treatment on blood granulocyte respiratory burst and locomotive responses were examined. Differences in baseline granulocyte function in patients as well as dose-related effects of IL-1 treatment were observed. Patients enrolled early in the trial (low-dose patients) had significantly lower locomotive responses before treatment than their paired controls; these low responses normalized after 5 days of continuous-infusion IL-1 treatment. Patients enrolled later (high-dose patients) had normal locomotive responses before treatment and IL-1 treatment was associated with suppression of responses to selected stimuli at the end of treatment. Pretreatment respiratory burst responses in both low-and high-dose patient groups were essentially normal, but the rates of granulocyte H₂O₂ production following phorbol myristate acetate stimulation became significantly less than control values at the end of treatment. In vitro exposure of either patient or control cells to 150 U/ml IL-1 did not alter their locomotive or respiratory burst responses, suggesting the observed in vivo effects were not mediated directly by IL-1. Treatment with IL-1 is associated with changes in ex vivo granulocyte function that are related to the IL-1 dose. Treatment with low doses of IL-1 may provide a means of normalizing abnormal polymorphonuclear leukocyte function in some patients with ovarian malignancies.     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

Effects of selective α1A-adrenoceptor antagonists on reperfusion arrhythmias in isolated rat hearts

Stimulation of ₁-adrenoceptors (AR) during ischaemia in the rat heart by exogenous phenylephrine exacerbates reperfusion arrhythmias, an effect apparently mediated by the _1A-AR subtype. We tested whether _1A-AR stimulation byendogenous catecholamines, released during ischaemia, could modulate reperfusion arrhythmias, using as pharmacological tools the selective _1A-AR antagonists abanoquil (UK52046) and WB4101. Isolated rat hearts (n=12/group) were subjected to dual coronary perfusion. After 15 min of aerobic perfusion of both coronary beds, abanoquil or WB4101 was infused selectively into the left coronary bed (LCB) for 5 min. The LCB was then subjected to 10 min of zero-flow ischaemia and 5 min of reperfusion. Effects on PR interval, width of the ventricular complex (QRST₉₀) and reperfusion arrhythmias were assessed. Abanoquil at concentrations of 0.03, 0.1 and 0.3 M tended to reduce the incidence of reperfusion-induced ventricular fibrillation (VF) in a dose-dependent manner from 75% in controls to 58, 33 and 25%, but this effects did not achieve statistical significance. Similarly, WB4101 at 0.1, 0.3 and 1 M also tended to reduce VF incidence from 67% in controls to 67, 42% and 33% (NS). The incidence of ventricular tachycardia (VT) was 100% in all groups and ECG parameters were not altered significantly by either drug. These results suggest that, in this denervated isolated heart preparation, _1A-AR stimulation during ischaemia by endogenous catecholamines does not significantly modulate reperfusion arrhythmias.     

1H-filtered correlation experiments for assignment and determination of coupling constants in backbone labelled proteins

The implementation of [¹³C,¹³C,¹⁵N,²H] labelled amino acids into proteins allows the acquisition of high resolution triple resonance experiments. We present for the first time resonance assignments facilitated by this new labelling strategy. The absence of ¹J_C,C couplings enables us to measure ¹J_C,C scalar and ¹D_C,C residual dipolar coupling constants using modified HNCA experiments which do not suffer from sensitivity losses characteristic for ¹³C constant time experiments.     

Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology

Summary We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage _a and decreased the fractional resistance of the apical membranef _R. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized _a and increasedf _R, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, _a depolarized andf _R decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of adrenergic receptors because it was reproduced by addition of the agonist phenylephrine, and blocked by the antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of ₁ adrenergic receptors. The acute adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both and adrenergic agents mediate the ion transport processes in canine tracheal epithelium. adrenergic agents have their primary effect on the apical Cl conductance, probably via an increase in cAMP. adrenergic agents exert their primary effect on the basolateral K conductance, possibly via an increase in cell Ca.     

A new α2HS-glycoprotein typing by isoelectric focusing

Summary Isoelectric focusing (pH 4.0–5.0) of serum ₂HS-glycoprotein on polyacrylamide gels has been found to be a useful tool in population genetics and forensic science. Using this method, we isolated three common types, ₂HS 1-1, ₂HS 2-1 and ₂HS 2-2, and showed that ₂HS types are determined by two autosomal codominant alleles, ₂ HS ¹ and ₂ HS ². The method is simple, fast and easy to perform. Results of typing for the two alleles, ₂ HS ¹ and ₂ HS ², are described for a Japanese population sample (n=1003).     

The comparative effects of IL-1 and TNF on AMN-anti-Ly 2.1 immunoconjugate therapy

The administration of mTNF and hIL-1 was investigated for their potential to increase the anti-tumor activity of AMN-anti-Ly-2.1 against the Ly-2.1⁺ murine thymoma ITT(1) 75 NS E3. Dose response studies using mTNF alone demonstrated a single 10g iv injection produced 30% inhibition in tumor size while 3 doses of 1g administered on alternative days produced 70% tumor inhibition. By contrast, hIL-1 was unable to significantly reduce E3 tumor size using single doses up to 10g or a total of 30g administered in 3 doses (iv or ip). However, intratumor injection of hIL-1 (20g injected in 2 doses) produced 20% inhibition in tumor size. Combination therapy using AMN immunoconjugates with mTNF showed enhanced antitumor activity compared to each agent alone. Biodistribution studies revealed that anti-tumor activity, was due to increased localization (2–3 fold) of AMN immunoconjugates in the presence of mTNF- whereas huIL-1 was without effect unless accompanying toxicity was seen. Clearly for this tumor, mTNF potentiated the effects of AMN immunoconjugates. Despite the shared biological properties of these cytokines, mTNF is superior to hIL- for augmenting drug immunoconjugate.Abbreviations AMN Aminopterin - CBF₁ (C57BL6xBALB/c)F₁ mice - DMSO Dimethyl sulfoxide - E3 ITT(1)75NS E3 - HAMA human-anti-mouse antibody - i.p. intraperitoneal(ly) - I.T. intratumor - i.v. intravenous(ly) - hIL-1 recombinant human Interleukin-1-alpha - MoAb monoclonal antibody - PBS phosphate buffered saline - SE standard error - s.c. subcutaneous(ly) - mTNF recombinant murine tumor necrosis factor-alpha     

Expression of neolactoglycolipids: sialosyl-, disialosyl-,O-acetyldisialosyl- and fucosyl- derivatives of neolactotetraosyl ceramide and neolactohexaosyl ceramide in the developing cerebral cortex and cerebellum

The following neolacto glycolipids were identified and their developmental expression was studied in the rat cerebral cortex and cerebellum: Fuc1-³IIInLcOse₄Cer,Fuc1-³VnLcOse₆Cer and (Fuc)₂1-³III,³VnLcOse₆Cer, as well as acidic glycolipids, NeuAc2-³IVnLcOse₄Cer [nLM1], (NeuAc)₂2-³IVnLcOse₄Cer [nLD1],O-acetyl (NeuAc)₂2-³IVnLcOse₄Cer [OAc-nLD1] and their higher neolactosaminyl homologues NeuAc2-³VInLcOse₆Cer [nHM1] and (NeuAc)₂2-³VInLcOse₆Cer [nHD1]. These glycolipids were expressed in the cerebral cortex only during embryonic stages and disappeared postnatally. This loss was ascribed to the down regulation of the synthesis of the key precursor LcOse₃Cer which is synthesized by the enzyme lactosylceramide:N-acetylglucosaminyl transferase. On the other hand in the cerebellum, these glycolipids increased with postnatal development due to increasing availability of LcOse₃Cer. In the cerebellum, only nLM1 and fucosyl-neolactoglycolipids declined after postnatal day 10–15, perhaps due to regulation by other glycosyltransferases. Also, in the cerebellum, nLD1 and nHD1 were shown to be specifically associated with Purkinje cells and their dendrites in the molecular layer and with their axon terminals in the deep cerebellar nuclei, similar to other neolactoglycolipids shown previously.     

Genetically determined differences in the response of alpha1-antitrypsin levels in human serum to typhoid vaccine

Summary Normal individuals and those homozygous and heterozygous for the gene which determines deficiency in serum ₁-antitrypsin (₁-at) were given an intravenous injection of typhoid vaccine. Following this injection the trypsin-inhibiting capacity of serum and the concentration of ₁-at increases markedly in normal individuals. Heterozygotes for the deficiency gene have an elevation of ₁-at and trypsin-inhibiting capacity of their serum but reach only 50 percent of the levels reached by homozygotes for the common gene (normal individuals). Their values are however temporarily in the range of normal men not given typhoid vaccine. Homozygotes for the deficiency gene show only a small elevation of ₁-at under these conditions. These findings demonstrate that (1) the increase in trypsin-inhibiting capacity of serum after an injection of typhoid vaccine is largely due to the elevation of the ₁-at concentration, and (2) the ₁-at deficiency gene inhibits the quantitative response of the ₁-at to such a stimulus.Supported in part by USPHS grant HE-06285 from the National Heart Institute.Some of these studies were carried out in the General Clinical Research Center Ward, U. C., FR-79, supported by the Division of Research Facilities and Resources, U.S. Public Health Service.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

P2Y6 nucleotide receptor activates PKC to protect 1321N1 astrocytoma cells against tumor necrosis factor-induced apoptosis

1. We recently reported that the activation by UDP of rat P2Y₆ nucleotide receptors expressed in 1321N1 astrocytoma cells protected them from TNF-induced apoptosis by suppressing activation of caspase 3 and 8. This study aims to characterize the involvement of intracellular signaling pathways, including kinases, involved in the antiapoptotic effect of UDP.2. Cell death was induced in 1321N1 astrocytoma cells permanently expressing the rat P2Y₆ receptor by exposure to TNF in the presence of cycloheximide. The apoptotic fraction was analyzed using flow cytometry.3. The activation of P2Y₆ receptors by UDP both protected the astrocytes from TNF- induced apoptosis and activated protein kinase C (PKC) isotypes. The phorbol ester PMA also activated PKC and protected the cells from TNF-induced cell death. The - and -isotypes of PKC were both activated in a persistent fashion upon 5-min exposure to either UDP (10 M) or the phorbol ester PMA (100 nM). The PKC isotype was markedly activated upon UDP treatment.4. The addition of PKC inhibitors, GF109203X or Gö6976, partially antagonized the protective effect of UDP and reduced the UDP-induced phosphorylation of extracellular signal-regulated protein kinases (Erk). The inhibitors of Erk, PD98,059 or U0126, antagonized UDP-induced protection.5. The antiapoptotic protein, Akt, was not affected by P2Y₆ receptor activation. Incubation of the astrocytes with calcium modifiers, BAPTA-AM or dantrolene, did not affect the UDP-induced protection from apoptosis.6. The addition of phospholipase C (PLC) inhibitors, D609 or U73122, partially antagonized both UDP-induced protection and PKC activation.7. Therefore, it is suggested that P2Y₆ receptors in 1321N1 cells, through coupling to PC-PLC and PI-PLC, activate PKC to protect against TNF -induced apoptosis, in which the activation of Erk is involved in part.     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

A solid-phase synthesis of three aza-, iminoaza- and reduced aza-peptides from the same precursor

Using the Boc-strategy, a step-by-step synthesis on the PAM solid supportof three aza-, iminoaza- and reduced aza-peptide homologues is described.From the same hydrazinocarbonyl peptide-PAM precursor, the coupling ofeither a Boc-amino acid or a Boc-amino aldehyde gives rise to an aza-peptideor an iminoaza-peptide containing theC-CO-NH-N-CO-NH-C orC-CH=N-N-CO-NH-C surrogate of the peptide motif, respectively. In situreduction of the latter by NaBH₃CN leads to a reducedaza-peptide containing theC-CH₂-NH-N-CO-NH-C moiety. The key step synthesis of thehydrazinocarbonyl peptide-PAM precursor is carried out by coupling on thegrowing peptide chain the N-Boc-aza-amino acid chloride obtained by theaction of triphosgene on the corresponding N-Boc-hydrazine. Thesemodifications have been introduced in position 1-2 of the YLGYLEQLLRbenzodiazepine-like decapeptide     

Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: Possible involvement of tyrosine kinase and protein kinase C

Monocytic U937 cells were differentiated into mature macrophages in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h at 37°C. We investigated the alterations in the expression of GTP-binding proteins that take place during differentiation of these cells. A 40 KDa -subunit of the inhibitory G-protein was identified by specific antibodies to G_i-1/2 and G_i-3 on Western blots and also by ADP-ribosylation catalyzed by pertussis toxin. The expression of the 40 KDa G_i subunit was increased 3.4 fold in differentiated cells. The expression of a 43 kDa G_s subunit identified by Western blotting using specific antibody to G_s and by ADP-ribosylation in the presence of cholera toxin was increased approximately 2 fold in differentiated cells. A faintly recognizable 46 KDa G_s subunit was also increased but to a lesser extent (1.3 fold). Small molecular weight GTP-binding proteins identified by [³⁵S]GTPS binding on nitrocellulose blots were also increased significantly. The PMA-induced expression of G_i-1/2 and G_s subunits was blocked to control level by both genistein and staurosporine, inhibitors of protein tyrosine kinase and protein kinase C, respectively. However, staurosporine was unable to block the PMA-induced expression of G_i-3; this was blocked only by genistein. These data suggest a role for tyrosine kinase and protein kinase C in the expression of G-proteins during differentiation of U937 cells.