Biomedical subjects

J E Schultz

Publications and source records attributed to J E Schultz.

At least 19 recordsLinked to original sources

PubMed1999

Expression of membrane-bound and cytosolic guanylyl cyclases in the rat inner ear.

Membrane-bound guanylyl cyclases (GCs) are peptide hormone receptors whereas the cytosolic isoforms are receptors for nitric oxide. In the inner ear, the membrane-bound GCs may be involved in the regulation of fluid homeostasis and the cytosolic forms possibly play a role in signal processing and regulation of local blood flow. In this comprehensive study, we examined, qualitatively and quantitatively, the transcription pattern of all known GC isoforms in the inner ear from rat by RT-PCR. The tissues used were endolymphatic sac, stria vascularis, organ of Corti, organ of Corti outer hair cells, cochlear nerve, Reissner's membrane, vestibular dark cells, and vestibular sensory cells. We show that multiple particulate (GC-A, GC-B, GC-D, GC-E, GC-F and GC-G) and several subunits of the heterodimeric cytosolic GCs (alpha1, alpha2, beta1 and beta2) are expressed, albeit at highly different levels. GC-C was not found. GC-A and the soluble subunits alpha1 and beta1 were transcribed ubiquitously. GC-B was present in all tissues except stria vascularis, which contained GC-A and traces of GC-E and GC-G. GC-B was by far the predominant membrane-bound isoform in the organ of Corti (86%), Reissner's membrane (75%) and the vestibulum (80%). Surprisingly, GC-E, a retinal isoform, was detected in significant amounts in the cochlear nerve (8%) and in the organ of Corti (4%). Although the cytosolic GC is a heterodimer composed of an alpha and a beta subunit, the mRNA transcription of these subunits was not stoichiometric. Particularly in the vestibulum, the transcription of the beta1 subunits was at least four-fold higher than of the alpha1 subunit. The data are compatible with earlier suggestions that membrane receptor GCs may be involved in the control of inner ear electrolyte and fluid composition whereas NO-stimulated GC isoforms mainly participate in the regulation of blood flow and supporting cell physiology.

Animals↗

PubMed1998

Functional characterization and localization of protein phosphatase type 2C from Paramecium.

We cloned a protein phosphatase 2C gene from Paramecium (PtPP2C), which codes for one of the smallest PP2C isoforms (Klumpp, S., Hanke, C., Donella-Deana, A., Beyer, A., Kellner, R., Pinna, L. A., and Schultz, J. E. (1994) J. Biol. Chem. 269, 32774-32780). After mutation of 9 ciliate Q codons (TAA) to CAA PtPP2C was expressed as an active protein in Escherichia coli. The catalytic core region contains 284 amino acids as defined by C- and N-terminal deletions. The C terminus from amino acid 200-300 of PP2C isoforms has only about 20% similarity. To demonstrate that the carboxy end is in fact needed for activity, we generated an enzymatically active PtPP2C containing a C-terminally located tobacco etch virus-protease site. Upon proteolytic truncation enzyme activity was lost, i.e. the C terminus of PP2C is indispensable for enzyme activity. During these experiments isoleucine 214 was fortuitously identified to be essential for PP2C catalysis. Mutation of the hydrophobic amino acid to glycine in the ciliate or bovine isoforms resulted in inactive protein. Because Ile214 is in a loop region without defined secondary structure, our data clearly go beyond the x-ray structure. The functional equivalence of the 180 amino acid long C terminus from the bovine PP2C with the 100 amino acid long carboxy end of the PtPP2C was demonstrated by producing an active chimera, i.e. the PP2C from Paramecium has no obvious regions which may be specifically involved in subcellular localization or substrate recognition. Using antibodies against recombinant PtPP2C we localized the enzyme by immunogold labeling in the cytosol and nucleus and very distinctly on the ciliary microtubule/dynein complex. The data suggest a role for PtPP2C in the regulation of dyneins, i.e. in cellular cargo transport and ciliary motility.

Amino Acid Sequence↗

PubMed1998

Ischemic preconditioning in the intact rat heart is mediated by delta1- but not mu- or kappa-opioid receptors.

BACKGROUND: Our laboratory has previously shown that delta-opioid receptors are involved in the cardioprotective effect of ischemic preconditioning in the rat heart. However, this class of receptors consists of two subtypes, delta1, and delta2, and mu- or kappa-opioid receptors may also exist in the heart. Therefore, the purpose of the present study was to test the hypothesis that ischemic preconditioning is mediated through stimulation of one or both delta-opioid receptor subtypes. METHODS AND RESULTS: Anesthetized, open chest, male Wistar rats were assigned to 1 of 14 groups. All animals were subjected to 30 minutes of occlusion and 2 hours of reperfusion. Ischemic preconditioning was elicited by three 5-minute occlusion periods interspersed with 5 minutes of reperfusion. Two doses of 7-benzylidenenaltrexone (BNTX; 1 and 3 mg/kg i.v.), a selective delta1-opioid receptor antagonist, or naltriben (NTB; 1 and 3 mg/kg i.v.), a selective delta2-opioid receptor antagonist, were given before ischemic preconditioning. To test for a role of mu-opioid receptors, rats were pretreated with beta-funaltrexamine (beta-FNA; 15 mg/kg s.c), an irreversible mu-opioid receptor antagonist, 24 hours before ischemic preconditioning or given the mu-opioid receptor agonist D-Ala,2N-Me-Phe,4glycerol5-enkephalin (DAMGO) as three 5-minute infusions (1, 10, and 100 microg/kg per infusion i.v., respectively) interspersed with 5-minute drug-free periods before the prolonged ischemic and reperfusion periods (lowDAMGO, medDAMGO, and hiDAMGO, respectively). The involvement of kappa-opioid receptors was tested by administering one of two doses of nor-binaltorphimine (nor-BNI; 1 and 5 mg/kg i.v.) before ischemic preconditioning. Infarct size (IS) as a percent of the area at risk (AAR) was measured by triphenyltetrazolium stain. Ischemic preconditioning markedly reduced IS/AAR (14+/-4%, P<.05) compared with control (55+/-4%). NTB, beta-FNA, and nor-BNI were unable to block the cardioprotective effect of ischemic preconditioning. In addition, DAMGO had no effect on IS/AAR. However, the high dose of BNTX (3 mg/kg i.v.) significantly attenuated the cardioprotective effect of ischemic preconditioning (39+/-5%; P<.05 versus control and ischemic preconditioning). CONCLUSIONS: These results indicate that delta1-opioid receptors play an important role in the cardioprotective effect of ischemic preconditioning in the rat heart.

Animals↗

PubMed1998

Terikalant, an inward-rectifier potassium channel blocker, does not abolish the cardioprotection induced by ischemic preconditioning in the rat.

Recent results have shown that the sulfonylurea receptor couples to several types of inward-rectifier potassium (KIR) channels, which suggests that sensitivity to blockade of a pathophysiological phenomenon such as ischemic preconditioning (PC) by glibenclamide may not be the result of this compound selectively blocking the ATP-sensitive potassium (KATP) channel. Therefore, to address this possibility, a role for myocardial KIR v KATP channels in ischemic PC was evaluated in the rat. To test this hypothesis, anesthetized, open-chest, male Wistar rats were assigned to one of seven experimental protocols. Animals assigned to group I (control) received 30 min of occlusion and 2 h of reperfusion. Ischemic PC was produced by 3x5-min occlusion and 2-h reperfusion periods (group II). Terikalant (TK), an inward-rectifier potassium channel blocker, was used to test the role of other K+ channels, most notably the KIR, in the cardioprotective effect of ischemic PC in the rat. TK was given at a dose of 3 mg/kg, i.v., 15 min before the prolonged occlusion and reperfusion periods (group III). In groups IV, V, and VI terikalant (1, 3 and 6 mg/kg, i.v.) was given 15 min before ischemic PC (lowTK+PC, medTK+PC and hiTK+PC, respectively). Group VII consisted of glibenclamide (0.3 mg/kg, i.v.) given 30 min prior to ischemic PC (GLY+PC). Infarct size (IS) as a percent of the area at risk (AAR) was measured using the histochemical stain, 2,3, 5-triphenyltetrazolium chloride. The average IS/AAR for the control was 49.9+/-2.1%. Ischemic PC markedly reduced infarct size (8.6+/-1. 8%; * P<0.05 v control). Terikalant (TK; 1, 3 and 6 mg/kg, i.v.) did not abolish the cardioprotective effect of ischemic PC at any dose (15.5+/-6.4, 16.4+/-5.2 and 8.8+/-1.6%, respectively; * P<0.05 v control). TK itself had no effect on infarct size. GLY completely abolished the cardioprotective effect of ischemic PC (48.2+/-6.4%). In addition, the high dose of TK significantly (P<0.05) increased the action potential duration at 50% repolarization from 48+/-3 to 64+/-4 ms and 30 microM of TK, a concentration which produced a 39% decrease in the inward-rectifier potassium channel current in isolated guinea-pig ventricular myocytes in the whole-cell patch-clamp mode did not block the increase in K ATP current produced by the KATP opener bimakalim (3 microM). These results demonstrate that although the myocardial KATP channel belongs to the K IR superfamily, the endogenous myocardial KIR channel does not mediate ischemic PC in the rat heart; however, the K ATP channel does mediate its cardioprotective effect.

ATP-Binding Cassette Transporters↗

PubMed1998

Pertussis toxin abolishes the cardioprotective effect of ischemic preconditioning in intact rat heart.

It has been previously demonstrated that Gi/o proteins are involved in ischemic preconditioning (IPC) in rabbits and dogs; however, there has been controversy as to the role of Gi/o proteins in IPC in in vivo rat infarct models. Therefore, the role of G proteins in the cardioprotective effect of IPC in a rat infarct model was reevaluated. Cardioprotection as indicated by infarct size (IS) as a percentage of the area at risk (IS/AAR) was determined by triphenyltetrazolium stain. The control group, which was subjected to 30 min of occlusion (Occ) and 2 h of reperfusion (Rep), had an IS/AAR of 46 +/- 6%. A single 5-min Occ followed by 10 min of Rep (1x PC) as well as three 5-min Occ periods interspersed with 5 min of Rep (3x PC) markedly reduced IS/AAR (6 +/- 1 and 8 +/- 1%, respectively). Pretreatment with pertussis toxin (10 microg/kg ip) for 48 h before 1x PC or 3x PC completely abolished their cardioprotective effects (46 +/- 5 and 38 +/- 4%, respectively). Pertussis toxin had no effect on IS/AAR and did not inactivate Gi/o proteins as assessed by an in vitro ADP-ribosylation assay of heart homogenates. These results demonstrate that the cardioprotective effect of IPC is mediated by a small subpopulation of Gi/o proteins in the intact rat heart.

Acetylcholine↗

PubMed1998

Pretreatment with tyrosine kinase inhibitors partially attenuates ischemic preconditioning in rat hearts.

Ischemic preconditioning (IPC) confers cardioprotection against a prolonged ischemic insult. Tyrosine kinase (TK) inhibitors have been shown to attenuate IPC; however, it is unclear whether TK is involved in the initiation of and/or the maintenance of this phenomenon. Thus the hypothesis that TK acts primarily during the initiation of IPC was examined in a rat model of myocardial infarction. Hearts were subjected to 30 min of coronary artery occlusion and 2 h of reperfusion. IPC was elicited by three 5-min occlusions interspersed with 5 min of reperfusion before the prolonged occlusion period. Genistein, a nonspecific TK inhibitor, was administered before or during the final 2 min of the first or third occlusion period of IPC. Daidzein, an inactive structural analog of genistein, and lavendustin A, a more specific TK inhibitor, were also tested in this model. IPC markedly reduced infarct size expressed as a percentage of the area at risk compared with control (56.3 +/- 2.8 to 7.1 +/- 2.0%). This cardioprotection was attenuated by genistein pretreatment (5 mg/kg: 34.7 +/- 2.2%, 10 mg/kg: 33.5 +/- 5.9%). However, genistein administered during the first or third occlusion period of IPC did not significantly attenuate cardioprotection (10.3 +/- 2.9% and 6.4 +/- 2.0%). Lavendustin A (1. 0 mg/kg) pretreatment also attenuated IPC (30.1 +/- 2.2%), whereas daidzein (5 mg/kg) had no effect (7.9 +/- 2.4%). These results suggest that activation of a TK is involved in the initiation but not the maintenance of IPC in the rat myocardium.

Animals↗

PubMed1998

Adenylyl cyclase type 7 is the predominant isoform in the bovine retinal pigment epithelium.

The retinal pigment epithelium (RPE) fulfills important supporting tasks to maintain the visual functions of the sensorineural retina. One major signalling mechanism by which adjacent tissues impinge on the RPE is the adenylyl cyclase (AC)/cAMP pathway. In the RPE, cAMP seems to modulate unique functions such as the phagocytosis of discs shed from the rod outer segments, transport of vitamin A or the ion and fluid control in the subretinal space. We analyzed the AC expression pattern in the retina and the RPE and found AC type 7 to be almost the only isoform expressed in the RPE. We cloned AC type 7 from a cDNA library established with fresh bovine RPE, expressed this isoform in eukaryotic cells and characterized some of its properties.

Adenylyl Cyclases↗

PubMed1997

The inhibition of cathepsin S by its propeptide--specificity and mechanism of action.

The interaction of human recombinant full-length cathepsin S propeptide (amino acids 16-114) with mature cysteine proteinases was studied with respect to selectivity and pH dependence. The inhibitory capacity was tested towards mature human recombinant cathepsin S, purified cathepsin L from rat and Paramecium tetraurelia, rat cathepsin B, human cathepsin H, and papain. The propeptide of cathepsin S strongly inhibited cathepsin S (Ki = 0.27 nM) and the two cathepsin L species (Ki = 0.36 nM) at neutral pH. Papain, and to a minor extent cathepsin H, hydrolyzed the propeptide of cathepsin S, leading to competition with the hydrolysis of the fluorogenic substrates in the respective assays. Cathepsin B activity was nearly unaffected up to micromolar propeptide concentrations in the assay. The inhibition of cathepsin-L-like peptidases was diminished with decreasing pH, probably due to dramatic changes in the conformation of the propeptide. This assumption was supported by far-ultraviolet CD spectroscopy and by the finding of rapid hydrolysis of the cathepsin S propeptide by cathepsin L at pH values less than 5.5.

Animals↗

PubMed1997

Hyperpolarization- and depolarization-activated Ca2+ currents in Paramecium trigger behavioral changes and cGMP formation independently.

Using 5% ethanol as a deciliating agent, 20 mm colchicine to prevent reciliation and 1 mm amiloride to affect ion fluxes in Paramecium we examined the compartmentation and function of Ca2+ fluxes employing the biosynthesis of cGMP and the stereotypic swimming behavior as indicators for Ca2+ entry. As a function of extracellular Ca2+ Paramecia responded to colchicine and amiloride with a short-lived ciliary augmentation (fast swimming) which indicated hyperpolarization, and formation of cGMP, i.e., the reported hyperpolarization-activated Ca2+ inward current in the somatic membrane is coupled to intracellular generation of cGMP. This is comparable to the coupling of the depolarization-activated, ciliary Ca2+ inward current and ciliary cGMP formation.Ethanol-deciliated cells and ethanol-treated, yet ciliated control cells did not respond to a depolarization with backward swimming or formation of cGMP. Both responses recovered with similar kinetics. A persistent effect of an ethanol exposure on the axonemal apparatus or on guanylyl cyclase activity of ciliated control cells was excluded using permeabilized cells and cell-free enzyme, respectively. Further, in the presence of 20 mm colchicine ethanol-treated cells only recovered the depolarization-dependent avoiding reaction whereas the formation of cGMP remained depressed, i.e., the drug dissected both responses. Similarly, ethanol exposure of Paramecia did not affect the fast swimming response towards the hyperpolarizing agent amiloride whereas the cGMP formation was abrogated and recovered over a period of 7 hr, i.e., amiloride dissected the hyperpolarization-elicited behavioral response from the intracellular cGMP formation. The data demonstrate that in Paramecium depolarization- and hyperpolarization-stimulated behavioral responses and cGMP formation are not coupled. The behavioral changes are triggered by smaller Ca2+ inward currents than the formation of intracellular cGMP.

Amiloride↗

PubMed1997

Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity.

PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.

Amino Acid Sequence↗

PubMed1997

The ischemia-selective KATP channel antagonist, 5-hydroxydecanoate, blocks ischemic preconditioning in the rat heart.

Although the KATP channel has been demonstrated to be involved in ischemic preconditioning (IPC) in most species, controversy still exists as to the role of this channel as a mediator of PC in the rat heart. Previously, the authors' laboratories have shown that glibenclamide blocks IPC in the intact rat heart, in a time-dependent manner; however, since glibenclamide has been shown to have non-selective effects unrelated to KATP channel blockade, a structurally dissimilar and ischemia-selective KATP channel blocker, 5-hydroxydecanoate (5-HD), was used to further elucidate the role of KATP channels in mediating IPC in the rat heart. Anesthetized, open-chested Sprague-Dawley rats were subjected to one of four protocols. In Group I, control (C), rats were subjected to 30 min of left coronary artery occlusion and 90 min of reperfusion. In Group II, IPC was elicited by 1 x 5 min occlusion followed by 10 min of reperfusion, prior to the 30 min occlusion and 90 min reperfusion periods, 5-HD (5 mg/kg, i.v.) was given 15 min prior to the 30 min occlusion period in non-preconditioned animals, or given 15 min prior to IPC (5-HD+IPC) (Groups III and IV, respectively). Infarct size (IS), as a percentage of the area at risk (AAR), was determined by triphenyltetrazolium staining. Ischemic preconditioning produced a marked reduction in infarct size (47.5 +/- 3.8% to 7.9 +/- 1.9%, *P < 0.01), which was completely abolished by 5-HD (50.5 +/- 2.6%). These data further suggest that the opening of KATP channels is an important component of IPC in the intact rat heart, similar to that observed in other species.

Adenosine Triphosphate↗

PubMed1997

Glibenclamide-induced blockade of ischemic preconditioning is time dependent in intact rat heart.

The ATP-sensitive potassium (KATP) channel has been demonstrated to be a potential mediator of ischemic preconditioning (PC) in most species, with the exception of the rat. This conclusion is based on the failure of the KATP channel antagonist glibenclamide (Glib) to block PC in this species. However, previous studies did not take into consideration the kinetic properties of Glib binding to its receptor in the rat myocardium. Therefore, the purpose of the present study was to test the hypothesis that ischemic PC is mediated by the myocardial KATP channel in the rat and that blockade of PC by Glib is a time-dependent phenomenon. Barbiturate-anesthetized, open-chest male Wistar rats were subjected to 30 min of occlusion and 2 h of reperfusion. Ischemic PC was elicited by three 5-min occlusion periods interspersed with 5 min of reperfusion. Infarct size (IS) as a percentage of the area at risk (AAR; IS/AAR) was determined with triphenyltetrazolium staining. PC and the KATP channel opener nicorandil (50 micrograms.kg-1.min-1 i.v.) resulted in marked reductions in IS/AAR from 53 +/- 3% to 8 +/- 1% and 17 +/- 5%, respectively (P < 0.05). Two doses of Glib (1 or 0.3 mg/kg i.v.) given 30 min before PC abolished the protective effect of PC; however, Glib (0.3 mg/kg i.v.) given 5 min before PC failed to block the effect. Glib administered 30 min before the 15-min nicorandil infusion blocked the cardioprotective effect of NC but did not block its transient hypotensive effect. These results demonstrate that the KATP channel is involved in ischemic PC in the intact rat heart and that the inhibitory effect of Glib to block PC is time dependent.

Animals↗

PubMed1996

Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide.

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatic characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His6 tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 microM.

Amino Acid Sequence↗

PubMed1996

Cloning and expression of a bovine adenylyl cyclase type VII specific to the retinal pigment epithelium.

A cDNA of a type 7 adenylyl cyclase isoform was cloned from a bovine retinal pigment epithelium cDNA library using oligonucleotides developed to conserved regions common to mammalian adenylyl cyclases. A 6.7 kb mRNA of very high abundance was uniquely present on Northern blots containing mRNA or total RNA from the pigment epithelium. This transcript was undetectable in all other tissues examined. The cDNA encoded a protein of 1,097 amino acids and exhibited the known doublet of 6 transmembrane-spanning regions in a hydrophobicity plot. The novel member of the type 7 adenylyl cyclase isoform was expressed in COS-1 cells. It was stimulated 10- and 20-fold by 10 microM GTP gamma S and 100 microM forskolin, respectively. The high expression rate exclusively in the retinal pigment epithelium suggests that this adenylyl cyclase isoform is involved in processes specific to this functionally exceedingly important subretinal cell layer.

Adenylyl Cyclases↗

PubMed1996

Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart.

Previous results from our laboratory have suggested that opioid receptors are involved in ischemic preconditioning (PC) in rat heart. Furthermore, other investigators have suggested that mu- and delta-opioid receptors mediate analgesia and hypoxic cerebral vasodilatation via opening of ATP-sensitive K+ (KATP) channels. Thus, the purpose of the present study was to test the hypothesis that activation of opioid receptors mimics the cardioprotective effect of ischemic PC and that this effect is produced by activation of KATP channels in the rat heart. Anesthetized open-chest Wistar rats were subjected to six different protocols. All groups were subjected to 30 minutes of occlusion and 2 hours of reperfusion. Ischemic PC was elicited by three 5-minute occlusion periods interspersed with 5 minutes of reperfusion. Similarly, morphine-induced PC was elicited by three 5-minute drug infusions (100 micrograms/kg i.v. ) interspersed with 5-minute drug-free periods before the prolonged 30-minute occlusion. Infarct size (IS) as a percentage of the area at risk (AAR) was determined by triphenyltetrazolium staining. Ischemic PC and morphine infusions resulted in similar reductions in IS/AAR from 56 +/- 5% to 11 +/- 3% and 12 +/- 5%, respectively (P < .05). Administration of glibenclamide (0.3 mg/kg i.v.), a KATP channel antagonist, or naloxone (3 mg/kg i.v.), a nonselective opioid receptor antagonist, both blocked the cardioprotective effects of morphine. These results indicate that opioid receptor stimulation results in a reduction in infarct size similar to that produced by ischemic PC. The effect of morphine is most likely mediated via an opioid receptor-KATP channel-linked mechanism in the rat heart, since glibenclamide abolished its protection.

Animals↗

PubMed1996

Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.

We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.

Amino Acid Sequence↗

PubMed1995

Evidence for involvement of opioid receptors in ischemic preconditioning in rat hearts.

The purpose of the present study was to investigate a possible role of opioid receptors in ischemic preconditioning (PC). To test this hypothesis, anesthetized, open-chest, male Wistar rats were subjected to five different protocols. In group I, the control group was subjected to 30 min of left coronary artery occlusion and 2 h of reperfusion. In group II, ischemic PC was elicited by three 5-min occlusion periods interspersed with 5 min of reperfusion. In group III, naloxone (NL, 3 mg/kg iv), a nonselective opioid antagonist, was given to nonpreconditioned rats 10 min before the 30-min occlusion period. Finally, NL was administered 10 min before preconditioning (NL + PC, group IV) or immediately after the last 5-min preconditioning period (PC + NL, group V). Infarct size (IS) as a percentage of the area at risk (AAR) (IS/AAR) was determined by 2,3,5-triphenyltetrazolium chloride staining. PC resulted in a marked reduction in myocardial IS from 45 +/- 5 to 8 +/- 1 (P < 0.05). NL treatment before or immediately after PC abolished this protective effect; however, NL had no effect on IS in non-PC rats. These results are the first to support the hypothesis that activation of opioid receptors may play an important role in ischemic PC in the rat myocardium.

Animals↗

PubMed1994

A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia. Purification, characterization, and cloning.

We isolated the first membrane-bound type 2C serine/threonine protein phosphatase from the ciliated protozoan Paramecium tetraurelia (PtPP2C). Three isozymes of 33, 32, and 31 kDa with a specific activity of 1 mumol.min-1.mg1 were purified from the ciliary membrane. All enzymatic properties including (a) insensitivity toward inhibitors of other protein phosphatase families such as okadaic acid and microcystin, (b) absolute requirement for divalent cations, and (c) substrate specificity tested with synthetic phosphopeptides were identical to mammalian PP2C enzymes and identified the PtPP2C as a canonical PP2C in spite of it being about 25% smaller. The NH2-terminal was blocked. Microsequencing of six tryptic peptides established a relationship to other PP2C enzymes. The PtPP2C gene was obtained using degenerate oligonucleotide primers and the polymerase chain reaction. The gene coded for a 33-kDa protein with 300 amino acids and had an (A+T) content of 62%, typical for this protozoan. Nine of 15 Gln residues are encoded by TAA, a universal stop codon which codes for Gln in Paramecium. A large truncation at the COOH-terminal is responsible for the smaller size of the PtPP2C. Only a single transcript of 1 kilobase was detected with a Northern blot indicating that the 32- and 31-kDa proteins were proteolytic products of the 33-kDa enzyme. Sequence comparisons with PP2C enzymes from rat, rabbit, yeast, Arabidopsis, and Leishmania defined a highly diverged enzyme family which shares three conserved domains, I, II, and III, accounting for about 25% of the primary structure. We demonstrated further that the distances between domains I/II and II/III are very similar in all PP2C enzymes (9-13 and 74-80 amino acids, respectively). However, the amino acid sequences of the spacer regions are unrelated. In addition, the COOH-terminal ends of 100-200 amino acids which comprise 30-50% of the enzyme, display no identity. A dendrogramm shows that PtPP2C surprisingly is most closely related to the mammalian PP2C, and enzymes from Leishmania, Arabidopsis, and yeast are more distant relatives.

Amino Acid Sequence↗