Robert P. Becker

Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors : The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells

Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P < .05; Kd, 2.3 +/- 0.8 and 5.9 +/- 1.3 nmol/L; n = 4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (Kd, 0.17 +/- 0.08 nmol/L; 8.1 +/- 0.9 fmol/10(6) cells). Enalaprilat decreased the IC50 of [Hyp3-Tyr(Me)8]BK, the BK analogue more resistant to ACE, from 3.2 +/- 0.8 to 0.41 +/- 0.16 nmol/L (P < .05, n = 3). The biphasic displacement curve of the binding of [3H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 mumol/L) potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P < .05, n = 7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B2 receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

Carboxypeptidase M in brain and peripheral nerves.

Abstract: Carboxypeptidase M (CPM), a plasma membrane‐bound enzyme, cleaves C‐terminal basic amino acids with a neutral pH optimum. We studied its distribution in human, baboon, and dog brain and in dog peripheral nerves. Areas were dissected, homogenized, centrifuged, and assayed for activity with dansyl‐Ala‐Arg. The corpus callosum and the pyramidal and optic tract were especially rich in CPM, whereas basal ganglia and cortex had low activity. The identity of the basic carboxypeptidase activity with CPM was shown by similarities in subcellular localization, membrane attachment, substrate hydrolysis, inhibition by a specific basic carboxypeptidase inhibitor, and cross‐reaction with anti‐human CPM antiserum. This antiserum immunoprecipitated an average of 85% of the activity in human and baboon brain and ∼66% in dog brain. CPM copurified with myelin extracted from the brain. Consistent with results obtained in placenta and cultured kidney cells, CPM in the brain appears to be membrane‐bound via a phosphatidylinositol glycan anchor. In the peripheral nerves, the specific activity in dog sciatic nerve and in vagus was high (98 and 149 nmol/h/mg of protein, respectively). In immunohistochemical studies, glia in the brain, which appear to be oligodendrocytes or astrocytes, and the outer aspects of myelin sheaths and Schwann cells in sciatic and vagus nerves were stained. We conclude that in some areas of the CNS and the PNS, CPM is closely associated with myelin and myelin‐forming cells. Northern blot analysis revealed the presence of mRNA coding for CPM in the brain, showing that the enzyme is indeed synthesized there.

Human ACE and bradykinin B2 receptors form a complex at the plasma membrane

To investigate how angiotensin I‐converting enzyme (ACE) inhibitors enhance the actions of bradykinin (BK) on B2 receptors independent of blocking BK inactivation, we expressed human somatic ACE and B2 receptors in CHO cells. Bradykinin and its ACE‐resistant analog were the receptor agonists. B2 fused with green fluorescent protein (GFP) and ACE were coprecipitated with antisera to GFP or ACE shown in Western blots. Immunohistochemistry of fixed cells localized ACE by red color and B2‐GFP by green. Yellow on plasma membranes of coexpressing cells also indicated enzyme‐receptor complex formation. Using ACE‐fused cyan fluorescent protein donor and B2‐fused yellow fluorescent protein (YFP) acceptor, we registered fluorescence resonance energy transfer (FRET) by the enhanced fluorescence of donor on acceptor photobleaching, establishing close (within 10 nm) positions of B2 receptors and ACE. Bradykinin stimulation cointernalized ACE and B2 receptors. We expressed ACE fused to N terminus of B2 receptors, anchoring only receptors to plasma membranes. Here, in contrast to cells, where both ACE and B2 receptors are separately anchored, ACE inhibitors neither enhance activation of chimeric B2 nor resensitize desensitized B2 receptors. Heterodimer formation between ACE and B2 receptors can be a mechanism for ACE inhibitors to augment kinin activity at cellular level.—Chen, Z., Deddish, P. A., Minshall, R. D., Becker, R. P., Erdös, E. G., Tan, F. Human ACE and bradykinin B2 receptors form a complex at the plasma membrane. FASEB J. 20, 2261–2270 (2006)

Carboxypeptidase M, a Glycosylphosphatidylinositol-anchored Protein, Is Localized on Both the Apical and Basolateral Domains of Polarized Madin-Darby Canine Kidney Cells

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor andN-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane γ-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4–8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells.

The Progression of Inflammation Parallels the Dermal Angiogenesis in a Keratin 14 IL-4-Transgenic Model of Atopic Dermatitis

The role angiogenesis plays in atopic dermatitis is not well understood. The authors previously demonstrated ultrastructurally dermal microvascular angiogenesis in the IL‐4‐transgenic mouse model of atopic dermatitis. Here, they determine the angiogenic factors involved in dermal microvascular angiogenesis, regulatory function of inflammatory cytokines on the VEGF‐A production, and microvascular permeability in this model. Computer‐assisted photometric analyses for immunofluorescence‐labeled CD31 demonstrated a progressive increase in blood vessel number, diameter, and percent dermal areas occupied by CD31+ vessels as the disease evolves in transgenic mice from before disease onset through early and late skin lesions. Similar findings were documented for VEGR2+ vessels. Quantification of skin angiogenic factor mRNAs showed progressive increase of transcripts of VEGF‐A, but not VEGF‐B, VEGF‐C, or VEGF‐D. ELISA showed a similar increase of VEGF‐A in the serum and skin of transgenic mice. IL‐6 and IFN‐γ stimulated VEGF‐A mRNA production in the skin and in primary keratinocytes of transgenic mice. Other skin angiogenic factors that increased included Ang‐1, Ang‐2, GBP‐1, and VE‐cadherin. Microvascular leakage began in the transgenic mouse skin before disease onset and peaked in the late stage. In conclusion, IL‐6 and IFN‐γ may play important roles in upregulation of VEGF‐A, along with other pro‐angiogenic factors, to induce dermal microvascular angiogenesis.

Theiler's Virus-Induced Intrinsic Apoptosis in M1-D Macrophages Is Bax Mediated and Restricts Virus Infectivity: a Mechanism for Persistence of a Cytolytic Virus

ABSTRACT Theilers murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus in the family Picornaviridae, is a highly cytolytic virus that produces necrotic death in rodent cells except for macrophages, which undergo apoptosis. In the present study we have analyzed the kinetics of BeAn virus infection in M1-D cells, in order to temporally relate virus replication to the apoptotic signaling events. Apoptosis was associated with early exponential virus growth from 1 to 12 h postinfection (p.i.); however, ≥80% of peak infectivity was lost by 16 to 24 h p.i. The pan-caspase inhibitor qVD-OPh led to significantly higher virus yields, while zVAD-fmk completely inhibited virus replication until 10 h p.i., precluding its assessment in apoptosis. In contrast, while zVAD-fmk significantly inhibited BeAn virus replication in BHK-21 cells at 12 and 16 h p.i., virus replication at these time points was not altered by qVD-OPh. Bax translocation into mitochondria, efflux of cytochrome c into the cytoplasm, and activation of caspases 9 and 3 between ∼8 and 12 h p.i. (all hallmarks of the intrinsic apoptotic pathway) were transiently inhibited by expression of Bcl-2, which is not expressed in M1-D cells. Thus, BeAn virus infection in M1-D macrophages, which restricts virus replication, provides a potential mechanism for modulating TMEV neurovirulence during persistence in the mouse central nervous system.

Angiogenesis: The Major Abnormality of the Keratin‐14 IL‐4 Transgenic Mouse Model of Atopic Dermatitis

Objective: Angiogenesis plays an important role in psoriasis, but its role in atopic dermatitis is unknown. The authors examined the dermal microvasculature of an IL‐4 transgenic mouse model of atopic dermatitis to determine whether angiogenesis was present.

Kallikreins when activating bradykinin B2 receptor induce its redistribution on plasma membrane

The bradykinin (BK) B2 receptor (R) is directly activated by kallikreins and other serine proteases independent of BK release. Both the Galpha(i) and Galpha(q) proteins are involved, shown by the release of arachidonic acid and [Ca2+]i elevation. Site-directed mutagenesis of the receptor and the lack of heterogeneous desensitization of the human B2R by the BK and kallikrein emphasize among others the differences between activation by the proteases and the peptide. To characterize further the mechanism thereby kallikreins activate and desensitize the B2R we investigated the distribution of the human B2R tagged with the green fluorescent protein (B2-GFP(Ct)) on the plasma membrane of stably transfected Chinese hamster ovary (CHO) cells. We visualized the movement of B2-GFP(Ct) R with confocal fluorescence microscopy after activation by BK or a by serine protease. Continued exposure of the cells to BK led to B2R internalization within 15-20 min. Porcine pancreatic and human recombinant tissue kallikreins induced a rapid definite redistribution of receptors on the plasma membrane within 5 min, prior to internalization. These effects of kallikrein were blocked by the B2R antagonist HOE 140 and by the kallikrein inhibitor, aprotinin. The B2R was also activated by endoproteinases LysC and ArgC and trypsin, but these enzymes did not induce redistribution, only internalization. In control experiments, kallikrein had no effect on cells transfected to stably express the angiotensin-converting enzyme-green fluorescent protein (GFP). Thus, kallikreins when activating the BK B2R also trigger its redistribution on plasma membrane.

Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring

Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.

Expression of Rat Kallikrein and Epithelial Polarity in Transfected Madin-Darby Canine Kidney Cells

Many properties of urinary kallikrein are well characterized, but the intracellular processing of prokallikrein and release by kidney cells have yet to be clarified. We report here on the synthesis of prokallikrein in Madin-Darby canine kidney (MDCK) cells transfected with rat submaxillary gland kallikrein cDNA and on its activation by MDCK cells and by an enriched liver Golgi membrane preparation. Transfected MDCK cells secreted only prokallikrein at both the apical and basolateral sides in about a 4:1 ratio, but cells transfected with kallikrein cDNA in reverse orientation or untreated cells released only traces of the enzyme. Prokallikrein, in culture medium or in homogenized MDCK cells, was fully activated by trypsin but activated only to 44% by thermolysin. Prokallikrein was synthesized and released into the medium at a high rate: the enzyme secreted by 5 x 10(6) cells in 24 hours cleaved 46 nmol/min D-Val-Leu-Arg-7-amino-4-methylcoumarin and liberated 63 ng/min bradykinin after activation. Immunocytology indicated the association of prokallikrein with the Golgi apparatus in the transfected cells. Antiserum to rat urinary kallikrein detected a single band in a Western blot of conditioned medium and also immunoprecipitated the enzyme. Aprotinin inhibited activated prokallikrein. Although MDCK cells released prokallikrein, their homogenates activated prokallikrein at both pH 5.5 and 7.5. Prokallikrein was also activated by a highly enriched liver Golgi membrane fraction and by an endoplasmic reticulum preparation, but the Golgi preparation was 38-fold more active. The activation was blocked significantly by inhibitors of serine proteases and less by cysteine protease inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)