Biomedical subjects

P A Insel

Publications and source records attributed to P A Insel.

At least 73 records · Page 4Linked to original sources

PubMed1996

Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas.

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN-enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).

ADP-Ribosylation Factors↗

PubMed1996

P2-purinoceptors utilize multiple signalling pathways in MDCK-D1 cells.

1. Madin-Darby canine kidney (MDCK) cells are a widely used model system for the study of epithelial cells. We have utilized a clonal variant, MDCK-D1, to examine signalling by P2-purinoceptors. 2. Several lines of evidence that lead us to conclude that MDCK-D1 cells co-express P2a- and P2y-purinoceptors and that both subtypes are linked to the release of arachidonic acid and metabolites (AA) include: (a) relative potency of nucleotide analogues in promoting AA release; (b) blockade by the antagonist suramin of response to the P2Y-selective agonist, 2-methylthio ATP (2-MT-ATP), but not to the P2a-selective agonist, UTP; and (c) additivity of response to 2-MT-ATP and UTP. AA release is a consequence of activation of phospholipase A2 (PLA2), most likely the 85 kDa cytosolic PLA2. 3. Treatment of MDCK-D1 cells with ATP, but not UTP, increases inositol 1,4,5-trisphosphate formation while both UTP and ATP increase phosphatidylcholine hydrolysis, ATP, UTP, and 2-MT-ATP can also stimulate phospholipase D activity. 4. Purine nucleotides increase cellular cAMP levels in MDCK-D1 cells in a manner that depends, at least in part, on activation of cyclooxygenase, since cAMP generation stimulated by ATP or UTP is inhibited by treatment of cells with indomethacin. Because cyclooxygenase-derived PGE2 can bind to prostaglandin receptors and stimulate synthesis of cAMP, nucleotides may raise cAMP in an autocrine or paracrine fashion. 5. Taken together, these results indicate that MDCK-D1 cells co-express P2a and P2y-purinoceptors and that these receptors utilize several mechanisms to regulate cell function, including activation of multiple phospholipases and autocrine/paracrine action of products.

Adenosine Triphosphate↗

PubMed1996

Cloning of GRK2 cDNA from S49 murine lymphoma cells.

Beta-adrenergic receptor kinase is a member of the G protein-linked receptor kinase (GRK1) family that elicits receptor desensitization. We have cloned GRK2 from S49 mouse lymphoma cells. The nucleotide sequences of rat GRK2 and GRK3 were aligned and conserved primers chosen for use in reverse transcription-polymerase chain reaction (RT-PCR) of S49 mRNA. Direct sequencing of the PCR fragment provided a rapid means to identify the expression of the GRK2 but not the GRK3 transcript in these cells. Unique expression of GRK2 in S49 cells was confirmed by Western blotting. Three additional pairs of primers were chosen from the rat GRK2 sequence to amplify overlapping regions that together encompassed the entire coding sequence. After attempts to ligate the four fragments of S49 cell GRK2 cDNA by using PCR proved unsuccessful, the intact cDNA was assembled by digesting the PCR products in the region of the overlaps and ligating them in a single step into pBlue-script SK(+).

Animals↗

PubMed1996

Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells.

We have characterized the signaling pathways of purinergic receptors present on the renal epithelial cell line, Madin-Darby canine kidney (MDCK, D1 subclone). Several lines of evidence are consistent with the conclusion that coexisting P2u and P2y receptors release arachidonic acid and metabolites (AA) from MDCK-D1 cells: 1) relative potencies of nucleotide analogues, 2) blockade of P2y agonist- but not P2u agonist-mediated release by suramin, and 3) additivity by 2-methylthio-ATP and UTP. Differences exist between the signaling pathways of the two receptors: pertussis toxin treatment partially inhibits P2u- but not P2y-mediated AA release, and P2y (but not P2u) receptors appear to stimulate D-myo-inositol 1,4,5-trisphosphate production. P2u-receptor occupancy results in both homologous and heterologous desensitization; P2y-receptor occupancy elicits only homologous desensitization. Both receptors stimulate phosphatidylcholine hydrolysis via phospholipase C activation. However, AA release appears to result from phospholipid deacylation by phospholipase A2 activation, rather than from alternate pathways that may include PLC activation. These results demonstrate for the first time that two subtypes of P2-purinergic receptors, P2u and P2y receptors, coexist on a single renal epithelium cell type and that these two receptor subtypes can promote AA release, probably via activation of PLA2.

Adenosine Triphosphate↗

PubMed1995

Nuclear phospholipase D in Madin-Darby canine kidney cells. Guanosine 5'-O-(thiotriphosphate)-stimulated activation is mediated by RhoA and is downstream of protein kinase C.

We have recently demonstrated the existence of an ATP-activated phospholipase D (PLD) in the nuclei of MDCK-D1 cells (Balboa, M. A., Balsinde, J., Dennis, E. A., and Insel, P. A. (1995) J. Biol. Chem. 270, 11738-11740). We have now found that nuclear PLD is synergistically activated by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and ATP in a time- and concentration-dependent manner, but these compounds do not alter the sensitivity of the enzyme to activation by Ca2+. The synergistic stimulation of PLD activity could be blocked by addition of the protein kinase C inhibitors chelerythrine and calphostin C. Stimulation by GTP gamma S was abolished by guanosine 5'-O-(2-thiodiphosphate). Incubation of isolated nuclei with Clostridium botulinum C3 exoenzyme inhibited the potentiating effect of GTP gamma S on ATP-dependent nuclear PLD activity. Moreover, use of the Rho GDP dissociation inhibitor to extract Rho family G proteins from cell nuclei also inhibits PLD activity. Western blot analyses of isolated nuclei revealed the presence of the small G protein RhoA, but not of RhoB or the ADP-ribosylation factor. GTP gamma S-stimulated ATP-dependent PLD activity could be reconstituted in Rho GDP dissociation inhibitor-washed nuclei by addition of recombinant prenylated RhoA, but not by addition of non-prenylated RhoA. Taken together, these results indicate that nuclear PLD activity is modulated via a RhoA-dependent activation that occurs downstream of protein kinase C. Nuclear PLD, which appears to be a previously unrecognized effector regulated by protein kinase C and G proteins, may be involved in the regulation of nuclear function or structure.

ADP Ribose Transferases↗

PubMed1995

Interaction of the protein nucleobindin with G alpha i2, as revealed by the yeast two-hybrid system.

The heterotrimeric G protein, G alpha i2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length G alpha i2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins. Nucleobindin appears to represent one of those proteins.

Animals↗

PubMed1995

Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes.

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5'-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.

Adenylyl Cyclases↗

PubMed1995

A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells.

Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.

Adenosine Triphosphate↗

PubMed1995

Developmental regulation of the alpha-subunit of Gq/G11 as determined by quantitative immunoblot analysis.

We have used immunoblot analysis with glutathione and S-transferase fusion proteins as protein standards to examine expression of G alpha q/11 in membrane preparations from different organs and to assess changes during normal development. Adult rat tissues expressed 4-25 pmol/mg membrane protein G alpha q/11, with neonates expressing two to three times higher levels in several tissues (brain, heart, liver, and kidney). By contrast, levels of Gaq/11 in lung were similar in adults and neonates. A Gaq-specific antiserum confirmed the high expression in neonatal brain, heart, liver and kidney. Our results show that Gaq is developmentally regulated in many rat tissues.

Amino Acid Sequence↗

PubMed1995

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling associated with chronic reduction in beta-adrenergic receptor agonist activation were found. First, there was coordinate downregulation of Gi alpha 2 and Gs alpha mRNA and protein expression in the left ventricle; reduced G-protein content was also found in the right atrium. Second, in the left ventricle, there was a twofold increase in beta-adrenergic receptor-dependent stimulation of adenylyl cyclase and a persistent high affinity state of the beta-adrenergic receptor. Finally, there was a reduction in left ventricular beta-adrenergic receptor kinase activity, suggesting a previously unrecognized association between the degree of adrenergic activation and myocardial beta-adrenergic receptor kinase expression. The heart appears to adapt in response to chronic beta-adrenergic receptor antagonist administration in a manner that would be expected to offset reduced agonist stimulation. The mechanisms for achieving this extend beyond beta-adrenergic receptor upregulation and include alterations in G-protein expression, beta-adrenergic receptor-Gs interaction, and myocardial beta-adrenergic receptor kinase activity.

Adaptation, Biological↗

PubMed1995

Colchicine and cytochalasin B enhance cyclic AMP accumulation via postreceptor actions.

The role of cytoskeletal microtubules and microfilaments in modulating cAMP generation in S49 lymphoma cells was investigated using the agents colchicine and cytochalasin B, respectively, which are known to disrupt these structures. A 1-hr pretreatment of S49 cells with 10 microM colchicine typically enhanced maximal isoproterenol-(beta-adrenergic receptor) stimulated cAMP accumulation by 100%, whereas cytochalasin B increased isoproterenol-stimulated cAMP by 30%. The combination of colchicine and cytochalasin B synergistically enhanced agonist-stimulated cAMP to 225% over control values. A synergistic increase in cAMP accumulation was also observed in cells treated with the agonist prostaglandin E1 or cholera toxin (which activates the stimulatory guanine nucleotide regulatory (Gs) protein). Colchicine and cytochalasin B did not ablate the inhibitory effects of somatostatin or the stimulatory effect of pertussis toxin treatment on beta-receptor-stimulated cAMP accumulation, indicating that these cytoskeletal disrupting agents do not enhance responsiveness in S49 cells via alterations in the inhibitory guanine nucleotide regulatory protein pathway. Moreover, colchicine, but not cytochalasin B treatment, enhances expression of isoproterenol-promoted 3H-forskolin binding in intact cells (a measure of Gs/adenylyl cyclase coupling). Thus, colchicine and cytochalasin B appear to enhance signaling in the Gs/adenylyl cyclase pathway by alterations of components distal to hormone receptors, most likely at the Gs protein and/or via cAMP generation. These results imply that microtubules and microfilaments can interact in the regulation of this pathway and that increases in cellular cAMP may contribute to the action of drugs that alter function of these cytoskeletal elements.

Adenylyl Cyclases↗

PubMed1994

Protein kinase C alpha mediates phospholipase D activation by nucleotides and phorbol ester in Madin-Darby canine kidney cells. Stimulation of phospholipase D is independent of activation of polyphosphoinositide-specific phospholipase C and phospholipase A2.

Protein kinase C (PKC) has been implicated in the activation of phospholipase D (PLD) in a number of systems. By antisense technology, we have "knocked out" alpha and beta isoforms of PKC to study the role of these isoforms in PLD activation in Madin-Darby canine kidney (MDCK) cells. To this end, we have studied PLD activation by phorbol 12-myristate 13-acetate (PMA), ATP, UTP, and 2-methylthio-ATP in cells labeled with [3H]palmitic acid. [3H]Phosphatidylethanol (PEt) production catalyzed by PLD in the presence of ethanol was time- and concentration-dependent in PMA- and nucleotide-stimulated cells. In Ca(2+)-free medium, [3H]PEt accumulation was diminished for all stimuli assayed. Treatment of cells with chelerythrine, an inhibitor of PKC, and phorbol ester down-regulation of PKC inhibited [3H]PEt production by both PMA and nucleotides. In cells transfected with antisense PKC alpha or both PKC alpha and PKC beta, PLD activation was inhibited by both PMA and nucleotides, whereas in cells transfected with antisense PKC beta, PLD activation was similar to that of control cells. Moreover, inhibition of polyphosphoinositide-specific PLC (by neomycin) or of release of arachidonic acid and arachidonic acid metabolites (by nordihydroguaiaretic acid or by indomethacin) failed to decrease [3H]PEt accumulation in PMA- and nucleotide-stimulated MDCK-D1 cells. From these data, we conclude that in MDCK-D1 cells PMA and nucleotide receptors utilize PKC alpha to regulate PLD activity and that PLD activation is independent of the activation of polyphosphoinositide-specific PLC and phospholipase A2-mediated release of arachidonic acid or arachidonic acid metabolites.

Animals↗

PubMed1994

Comparison of cloned and pharmacologically defined rat tissue alpha 1-adrenoceptor subtypes.

Multiple alpha 1-adrenoceptor subtypes have been defined by pharmacological and receptor cloning techniques, but the precise alignment of cloned and pharmacologically-defined subtypes is still unclear. We have compared the affinities of 8 subtype-selective compounds at three cloned alpha 1-adrenoceptor subtypes (rat alpha 1B, bovine alpha 1C, rat alpha 1A/D) with those previously determined by the same methods in rat spleen, cerebral cortex, and kidney (Naunyn-Schmiedeberg's Arch. Pharmacol. 348: 385-395, 1993). Among all compounds tested to date at cloned alpha 1-adrenoceptor subtypes (+)-tamsulosin appears to be the most selective with a rank order of potency alpha 1C > alpha 1A/D > or = alpha 1B. Affinities for the alpha 1A-selective 5-methyl-urapidil, methoxamine, oxymetazoline, phentolamine and (-)- and (+)-tamsulosin and for noradrenaline and SDZ NVI-085 at the splenic alpha 1B-adrenoceptors and at their low affinity sites in cerebral cortex and kidney correlated best with those at the cloned alpha 1B-adrenoceptor. Affinities of these drugs at their high affinity sites in cerebral cortex (pharmacologically-defined alpha 1A-adrenoceptor) were matched best by those at the cloned alpha 1C-adrenoceptor. Rat kidney appears to contain two chloroethylclonidine-resistant alpha 1-adrenoceptor subtypes one of which is similar to the cloned alpha 1C- and one to the cloned alpha 1A/D-adrenoceptor. We conclude that the cloned alpha 1B-adrenoceptor is the genetic correlate of the pharmacologically-defined alpha 1B-adrenoceptor. An alpha 1-adrenoceptor subtype corresponding to the cloned alpha 1A/D-adrenoceptor appears to exist in rat kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Is SDZ NVI-085 an alpha 1-adrenoceptor subtype-selective agonist?

SDZ NVI-085 has been proposed to be a centrally acting agonist with selectivity for some alpha 1-adrenoceptor subtypes. We have investigated its selectivity and efficacy at alpha 1-adrenoceptor subtypes in rat tissues and at cloned subtypes. SDZ NVI-085 had higher affinity for chloro-ethylclonidine-insensitive (alpha 1A-like) than for -sensitive (alpha 1B) alpha 1-adrenoceptors in rat kidney but not in cerebral cortex. SDZ NVI-085 recognized cloned alpha 1-adrenoceptor subtypes expressed in COS cells with the order of potency bovine alpha 1C > rat alpha 1A/D > rat alpha 1B. Relative to 100 microM noradrenaline, SDZ NVI-085 was only a partial agonist for stimulation of inositol phosphate formation in rat kidney and inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated tissue. We conclude that SDZ NVI-085 discriminates among multiple alpha 1-adrenoceptor subtypes and is a partial agonist at rat renal alpha 1A- and alpha 1B-adrenoceptors.

Adrenergic alpha-Agonists↗

PubMed1994

Immunoelectron microscopic identification of cytoplasmic and nuclear Gs alpha in S49 lymphoma cells.

The subcellular distribution of Gs alpha was characterized in S49 lymphoma cells with two polyclonal antisera directed against specific COOH- and NH2-terminal epitopes. Nonspecific binding was determined in each subcellular compartment by incubating cyc- S49 cells, known to be deficient in Gs alpha and its mRNA, with primary and secondary antisera. Small proportions of total specific binding sites were localized to the plasmalemma as well as the nuclear envelope. Because of their small size, these compartments contained a high concentration of Gs alpha. However, most of the specific binding sites were found in nonstructured cytoplasm and within the nucleus. Specific binding was abolished or significantly reduced by preincubating primary antisera with their peptide immunogens but not with an irrelevant peptide. Intracellular Gs alpha immunoreactive binding sites did not colocalize with gold-conjugated transferrin in cells preincubated with this ligand to mark a classical endocytotic pathway. The intracellular and intranuclear location of Gs alpha was confirmed with confocal microscopy of S49 cells immunostained with specific primary and fluorescently labeled secondary antibodies. Gs alpha was also detected with immunoblots of proteins extracted from purified S49 cell nuclei. Thus, Gs alpha is abundantly distributed in intracellular and intranuclear sites in S49 cells and occurs in loci distinct from organelles of the transferrin pathway. The substantial intracellular distribution of Gs alpha suggests that Gs may subserve intracellular and, perhaps, intranuclear functions that may be important in proliferating cells.

Animals↗

PubMed1994

Characterization of the alpha-1 adrenoceptor subtype mediating [3H]-arachidonic acid release and calcium mobilization in Madin-Darby canine kidney cells.

The authors characterized the alpha-1 adrenoceptor subtype located on Madin-Darby canine kidney (MDCK) cells by measuring norepinephrine-mediated [3H]-arachidonic acid release and Ca++ mobilization in fura 2-loaded cells. In both assays, prazosin and chloroethylclonidine acted as unsurmountable antagonists, whereas 5-methyl-urapidil acted as a competitive antagonist with pA2 estimates of 7.3 (arachidonic acid assay) and 7.7 (Ca++ assay). Competitive antagonism toward arachidonic acid release was also obtained with the following alpha-1 adrenoceptor antagonists (pA2): (+)-niguldipine (7.6), 2-(2,6-dimethoxy-phenoxy-yethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB 4101; 8.3), phentolamine (7.6) and oxymetazoline (6.4). Arachidonic acid release by norepinephrine was abolished in the absence of extracellular Ca++ and was antagonized by 1-[beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) but was insensitive to antagonism by L-type Ca++ channel antagonists and La . Norepinephrine-mediated increases in intracellular Ca++ consisted of two distinct phases: a transient phase followed by a sustained plateau. The transient phase was resistant to EGTA, whereas the plateau phase was abolished by EGTA. Potassium chloride did not evoke a response in either assay. Northern blot analysis demonstrated that MDCK cells express alpha-1B adrenoceptor messenger RNA. It was concluded that typical alpha-1B adrenoceptors mediate responses to norepinephrine in MDCK cells and that these receptors couple to both intracellular Ca++ release and Ca++ influx by a voltage-independent mechanism. This influx pathway is insensitive to L-type Ca++ channel antagonists but is antagonized by the receptor-operated Ca++ channel antagonist SK&F 96365.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed1993

Inhibition of expression of protein kinase C alpha by antisense cDNA inhibits phorbol ester-mediated arachidonate release.

A major unresolved issue in the area of signal transduction relates to the role of particular isoforms of protein kinase C (PKC) in mediating cellular responses subsequent to activation of that enzyme. We have addressed this issue by the use of antisense technology. We have stably transfected Madin-Darby canine kidney cells with antisense PKC alpha, PKC beta, or both PKC alpha and -beta cDNAs. The transfected cDNA was integrated and expressed. We have isolated cells in which expression of PKC alpha is inhibited. In cells transfected with antisense PKC alpha or both PKC alpha and -beta, phorbol ester-stimulated release of arachidonate and its metabolites was inhibited, whereas in cells transfected with antisense PKC beta cDNA alone, phorbol ester-stimulated arachidonate release was not significantly different from control cells. We thus demonstrate the use of a novel technique to inhibit PKC isoform expression. We show that inhibition of expression of PKC alpha causes a loss in phospholipase A2-mediated arachidonate release. Antisense-inhibited expression of PKC isoforms may provide a useful approach to define additional functions of particular PKC isoforms.

Animals↗

PubMed1993

Alteration in Gs-mediated signal transduction in S49 lymphoma cells treated with inhibitors of microtubules.

We have assessed the possible interaction between the microtubular component of the cytoskeleton and signal transducing GTP-binding (G) proteins by examining the ability of colchicine and vinblastine (two microtubule disrupters) to alter Gs and Gi protein activity in S49 lymphoma cells. Treatment of wild type S49 cells with cholchicine and vinblastine increased beta-adrenergic agonist- and prostaglandin (PG) E1-stimulated formation of cAMP. The microtubular inhibitor nocodazole also enhanced isoproterenol-stimulated cAMP accumulation, whereas the inactive analog of colchicine, beta-lumicolchicine, did not have this action. Based on data obtained with wild type, cyc-, and UNC S49 cells, we determined that enhancement in cyclic AMP accumulation is proximal to the catalytic (C) unit of adenylylcyclase, distal to hormone receptors, and seems to be located on Gs. Treatment with colchicine increased guanosine 5'-(gamma-thio)triphosphate-stimulated accumulation of cAMP in permeabilized wild type cells. The increase in activity of Gs appeared not to result from a change in the intracellular concentration of GTP. Treatment of cells with colchicine or vinblastine also increased the amount of the alpha s-C complex, as assessed by the binding of [3H]forskolin to intact cells at 37 degrees C. In contrast to the observed effect on Gs, treatment of wild type S49 cells with colchicine failed to modify the degree of inhibition of cAMP formation produced by somatostatin, which acts via the activation of Gi. These data suggest that microtubules regulate the ability of Gs to interact with and activate the catalyst of adenylylcyclase.

Adenylyl Cyclases↗