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Biomedical subjects

M D Hollenberg

Publications and source records attributed to M D Hollenberg.

At least 37 records · Page 2Linked to original sources
PubMed2000

Proteinase-activated receptor-2 (PAR-2): regulation of salivary and pancreatic exocrine secretion in vivo in rats and mice.

Proteinase-activated receptor-2 (PAR-2) is expressed throughout the gastrointestinal tract including the pancreas, and may be involved in digestive functions. The aim of our study was to evaluate a potential role for PAR-2 in regulating salivary and pancreatic exocrine secretion in vivo. PAR-2-activating peptides (PAR-2-APs), but not selective PAR-1-APs, administered intravenously, increased salivary secretion in the mouse or rat; this effect of the PAR-2-APs was unaffected by atropine, phentolamine, propranolol or indomethacin. Secretion (amylase) by rat parotid gland slices in vitro was also stimulated by PAR-2-APs and trypsin, but not by activation of other PARs. PAR-2-APs, administered to rats in vivo, caused a prompt effect on pancreatic exocrine secretion. PAR-2 mRNA, known to be present in pancreatic tissue, was also detected in parotid tissue. Our results indicate that in addition to a potential role in regulating cardiovascular and respiratory functions, PAR-2 may also play a general role in vivo for the direct regulation of glandular exocrine secretion.

Animals↗
PubMed2000

Proteinase-activated receptor-2-mediated matrix metalloproteinase-9 release from airway epithelial cells.

BACKGROUND: Matrix metalloproteinases (MMPs) digest extracellular matrix components and might be important mediators of tissue remodeling. Proteinase activated receptor-2 (PAR-2) is expressed in a variety of cell types including epithelial cells. PAR-2 receptors are activated by serine proteases such as trypsin and mast cell tryptase and have been implicated in inflammation. OBJECTIVE: To study the effects of PAR-2-mediated airway epithelial cell activation on the production of MMP-9. METHODS: A specific PAR-2-activating peptide and trypsin were used to activate the human airway epithelial cell line A549 as well as primary cultures of small airway epithelial cells (SAEC). MMP-2 and MMP-9 messenger RNA and enzymatic activity were evaluated by RT-PCR and gelatin zymography, respectively. RESULTS: PAR-2-activating peptides upregulated MMP-9 mRNA expression and release of MMP-9 enzymatic activity from airway epithelial cells but had no effect on MMP-2 production. Dexamethasone and budesonide (10(-6) to 10(-10) mmol) inhibited PAR-2-mediated MMP-9 release. Pretreatment with indomethacin indicated that MMP-9 release was not prostaglandin dependent. Inhibitors of the MAP kinase MEK- 1, and NFkappaB showed that both pathways are important for PAR-2-mediated MMP-9 release. Trypsin, a physiologic PAR-2 activator, upregulated MMP-9 but also MMP-2 release from airway epithelial cells. CONCLUSION: PAR-2 receptors appear to play an important role in the regulation of MMP-9 release from airway epithelial cells. As such, these receptors may be critical elements in tissue remodeling in asthma and other inflammatory conditions in the airways.

Adrenal Cortex Hormones↗
PubMed2000

Proteinase-activated receptors (PARs): activation of PAR1 and PAR2 by a proteolytic fragment of the neuronal growth associated protein B-50/GAP-43.

The neuronal growth associated protein B-50/GAP-43 has been localized in synaptosomes both as an intact protein and as a partial proteolysis product (termed B-60) that has an N-terminal sequence SFRGHITR.... Because of the relationship of this amino acid sequence to those of the tethered ligand for the human proteinase activated receptors PAR1 (SFLLRN...) and PAR2 (SLIGKV...), we wished to determine whether the B-50/GAP-43-derived proteolytic fragment SFRGHITR (SFR(B60)) might function as a PAR-activating peptide (PAR-AP) to stimulate either PAR1 or PAR2. With the use of a newly developed PAR1/PAR2 receptor activation-desensitization assay, employing PAR1/PAR2-bearing cultured human embryonic kidney (HEK293) cells, we found that SFR(B60) could activate both PAR1 and PAR2 so as to elevate intracellular calcium with EC50 values of approximately 200 and 50 microM, respectively. We also showed that trypsin can rapidly degrade B-50 to smaller fragments that would include the sequence SFR(B60). Because PAR1 and PAR2 are present on neurones, our data raise the possibility that in certain circumstances in vivo, B-50/GAP-43 may play a signalling role by serving as a precursor for proteolytically generated PAR-activating peptides.

Amino Acid Sequence↗
PubMed2000

Interleukin-1beta, Src- and non-Src tyrosine kinases, and nitric oxide synthase induction in rat aorta in vitro.

We studied the potential roles for endogenous interleukin-1beta (IL-1beta) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1beta augmented, whereas the IL-1beta receptor antagonist IL-1ra blocked, spontaneous iNOS induction. Furthermore, increases in IL-1beta mRNA preceded those of iNOS mRNA. Mitogen-activated protein kinase kinase and phosphatidyl inositol 3' kinase inhibition did not block iNOS induction, whereas nuclear factor kappaB inhibition did. The sarcoma virus tyrosine kinase (Src) family-selective inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) blocked the upregulation of IL-1beta mRNA and the subsequent induction of iNOS but not the induction of iNOS stimulated by exogenously added IL-1beta. In contrast, the non-Src inhibitors TP 47/AG 213 and genistein and the tyrosine phosphatase inhibitor vanadate did not affect the spontaneous upregulation of IL-1beta mRNA but blocked both the IL-1beta-mediated and spontaneous induction of iNOS. We conclude that 1) the upregulation of tissue IL-1beta, via a signaling pathway involving a Src family kinase, plays a key role in rat vascular iNOS induction and 2) non-Src tyrosine kinases play roles downstream from IL-1beta for iNOS induction.

Animals↗
PubMed2000

Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2.

Mast cells play a potentially important role in fibroproliferative diseases, releasing mediators including tryptase that are capable of stimulating fibroblast proliferation and procollagen synthesis. The mechanism by which tryptase stimulates fibroblast proliferation is unclear, although recent studies suggest it can activate protease-activated receptor (PAR)-2. We therefore investigated the role of PAR-2 in tryptase-induced proliferation of human fetal lung and adult lung parenchymal and airway fibroblasts and, for comparative purposes, adult dermal fibroblasts. Tryptase (0.7-70 mU/ml) induced concentration-dependent increases in proliferation of all fibroblasts studied. Antipain, bis(5-amidino-2-benzimidazolyl)methane, and benzamidine inhibited tryptase-induced fibroblast proliferation, demonstrating that proteolytic activity is required for the proliferative effects of tryptase. RT-PCR demonstrated the presence of PAR-2 mRNA, and immunohistochemical staining localized PAR-2 to the cell surface of lung fibroblasts. In addition, specific PAR-2 activating peptides, SLIGKV and SLIGRL, mimicked the proliferative effects of tryptase. In contrast, human dermal fibroblasts only weakly stained with the PAR-2 antibody, PAR-2 mRNA was almost undetectable, and fibroblasts did not respond to PAR-2 activating peptides. These results suggest that tryptase induces lung, but not dermal, fibroblast proliferation via activation of PAR-2 and are consistent with the hypothesis that the release of tryptase from activated mast cells may play an important role in the fibroproliferative response observed in asthma, chronic obstructive pulmonary disease, and patients with pulmonary fibrosis.

Adult↗
PubMed1999

Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2.

1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Animals↗
PubMed1999

Pro- and anti-inflammatory actions of thrombin: a distinct role for proteinase-activated receptor-1 (PAR1).

1. Thrombin has well characterized pro-inflammatory actions that have recently been suggested to occur via activation of its receptor, proteinase-activated receptor-1 (PAR1). 2. In the present study, we have compared the effects of thrombin to those of two peptides that selectively activate the PAR1 receptor, in a rat hindpaw oedema model. We have also examined whether or not thrombin can exert anti-inflammatory activity in this model. 3. Both thrombin and the two PAR1 activating peptides induced significant oedema in the rat hindpaw following subplantar injection. 4. The oedema induced by thrombin was abolished by pre-incubation with hirudin, and was markedly reduced in rats in which mast cells were depleted through treatment with compound 48/80 and in rats pretreated with indomethacin. In contrast, administration of the PAR1 activating peptides produced an oedema response that was not reduced by indomethacin and was only slightly reduced in rats pretreated with compound 48/80. 5. Co-administration of thrombin together with a PAR1 activating receptor resulted in a significantly smaller oedema response than that seen with the PAR1 activating peptide alone. This anti-inflammatory effect of thrombin was abolished by pre-incubation with hirudin. 6. These results demonstrate that the pro-inflammatory effects of thrombin occur through a mast-cell dependent mechanism that is, at least in part, independent of activation of the PAR1 receptor. Moreover, thrombin is able to exert anti-inflammatory effects that are also unrelated to the activation of PAR1.

Animals↗
PubMed1999

Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides in the rat paw.

In the present study, we have observed the development of an inflammatory reaction in the rat hindpaw, following the injection of specific agonists of PAR2 (two PAR2 activating peptides). This inflammation was characterized by oedema and granulocyte infiltration. Two selective PAR2 activating peptides, SLGRL-NH2 and trans-cinnamoyl-LIGRLO-NH2 induced significant oedema in the rat hindpaw from 1-6 h following subplantar injection. Six hours after the PAR2-activating peptide injection, the paw tissues showed a complete disruption of tissue architecture along with an inflammatory cell infiltrate. In the inflamed paw, PAR2-immunoreactivity was expressed on endothelial cells as well as on the infiltrating inflammatory cells. The oedema induced by the injection of the two PAR2 activating peptides was slightly reduced in rats pre-treated with compound 48/80, but was not modified by pre-treatment of rats with cromolyn, a mast cell stabilizer. Pre-treatment of rats with a cyclo-oxygenase inhibitor (indomethacin) or a nitric oxide synthase inhibitor (L-N(omega)-nitro-L-arginine methyl ester) had no effect on the oedema induced by the PAR2-activating peptides. These results demonstrate that the administration of PAR2-activating peptides into the rat paw induced an acute inflammatory response characterized by a persistent oedema (at least 6 h) and granulocyte infiltration. The PAR2-induced inflammatory response occurred through a mechanism largely independent of mast cell activation, and of the production of prostanoids and nitric oxide.

Animals↗
PubMed1999

Proteinase activated receptor 2: Role of extracellular loop 2 for ligand-mediated activation.

1. Rat proteinase-activated receptor-2 (PAR2) variants were stably expressed in rat KNRK cells: (a) wild-type (wt) - PAR2; (b) PAR2PRR, with the extracellular loop 2 (EL-2) sequence P231E232E233mutated to PRR and (c) PAR2NET, with the EL-2 sequence, PEEV changed to NETL. Cell lines were evaluated for their sensitivity (calcium signalling) towards trypsin and the receptor-activating peptides, SLIGRL-NH2, SLIGEL-NH2, trans-cinnamoyl(tc)-LIGRLO-NH2, and SFLLR-NH2. 2. SLIGEL-NH2 exhibited low potency (1 : 200 relative to SLIGRL-NH2) in wild-type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET. 3. In PAR2PRR, the potencies of SLIGRL-NH2, tc-LIGRLO-NH2, and SFLLR-NH2 were decreased by 80 - 100 fold. But, the potency of trypsin was decreased by only 7 fold. 4. In PAR2NET, highly homologous in EL-2 with proteinase-activated receptor-1 (PAR1), the potency of the PAR1-derived peptide, SFLLR-NH2, was reduced by 100 fold compared with wt-PAR2, whereas the potency of the PAR2-derived AP, SLIGRL-NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR2NET was almost the same as in wt-PAR2. 5. We conclude that the acidic EL-2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity. 6. The data obtained with the PEEV-->NETL mutation suggested: (a) that SLIGRL-NH2 and SFLLR-NH2 interact in a distinct manner with PAR2 and (b) that SFLLR-NH2 may interact differently with PAR2 than it does with PAR1. 7 The differential reductions in the potencies of SLIGRL-NH2, compared with trypsin in the PAR2PRR and PAR2NET cell lines point to differences between the interactions of the trypsin-revealed tethered ligand and the free receptor-activating peptide with PAR2.

Amino Acid Sequence↗
PubMed1999

Prevention of encephalomyocarditis virus-induced diabetes in mice by inhibition of the tyrosine kinase signalling pathway and subsequent suppression of nitric oxide production in macrophages.

Macrophages comprise the major population of cells infiltrating pancreatic islets during the early stages of infection in DBA/2 mice by the D variant of encephalomyocarditis virus (EMC-D virus). Inactivation of macrophages prior to viral infection almost completely prevents EMC-D virus-induced diabetes. This investigation was initiated to determine whether a tyrosine kinase signalling pathway might be involved in the activation of macrophages by EMC-D virus infection and whether tyrosine kinase inhibitors might, therefore, abrogate EMC-D virus-induced diabetes in vivo. When isolated macrophages were infected with EMC-D virus, inducible nitric oxide synthase mRNA was expressed and nitric oxide was subsequently produced. Treatment of macrophages with the tyrosine kinase inhibitor tyrphostin AG126, but not tyrphostin AG556, prior to EMC-D virus infection blocked the production of nitric oxide. The infection of macrophages with EMC-D virus also resulted in the activation of the mitogen-activated protein kinases (MAPKs) p42(MAPK/ERK2)/p44(MAPK/ERK1), p38(MAPK), and p46/p54(JNK). In accord with the greater potency of AG126 than of AG556 in blocking EMC-D virus-mediated macrophage activation, the incidence of diabetes in EMC-D virus-infected mice treated with AG126 (25%) was much lower than that in AG556-treated (75%) or vehicle-treated (88%) control mice. We conclude that EMC-D virus-induced activation of macrophages resulting in macrophage-mediated beta-cell destruction can be prevented by the inhibition of a tyrosine kinase signalling pathway involved in macrophage activation.

Animals↗
PubMed1999

[Physiology of protease-activated receptors (PARs): involvement of PARs in digestive functions].

The protease-activated receptor (PAR), a G protein-coupled receptor present on cell surface, mediates cellular actions of extracellular proteases. Proteases cleave the extracellular N-terminal of PAR molecules at a specific site, unmasking and exposing a novel N-terminal, a tethered ligand, that binds to the body of receptor molecules resulting in receptor activation. Amongst four distinct PARs that have been cloned, PARs 1, 3 and 4 are activated by thrombin, but PAR-2 is activated by trypsin or mast cell tryptase. Human platelets express two distinct thrombin receptors, PAR-1 and PAR-4, while murine platelets express PAR-3 and PAR-4. Apart from roles of PARs in platelet activation, PARs are distributed to a number of organs in various species, predicting their physiological importance. We have been evaluating agonists specific for each PAR, using multiple procedures including a HEK cell calcium signal receptor desensitization assay. Using specific agonists that we developed, we found the following: 1) the salivary glands express PAR-2 mRNA and secret saliva in response to PAR-2 activation; 2) pancreatic juice secretion occurs following in vivo PAR-2 activation; 3) PAR-1 and PAR-2 modulate duodenal motility. Collectively, PAR plays various physiological and/or pathophysiological roles, especially in the digestive systems, and could be a novel target for drug development.

Animals↗
PubMed1999

Differential induction of nitric oxide synthase in rat gastric and vascular smooth muscle tissue: distinct tissue distribution and distinctive signaling pathways.

In rat aorta rings (RA) and in gastric circular muscle (CM) and gastric longitudinal muscle (LM) preparations maintained in vitro, inducible nitric oxide synthase (iNOS) induction was monitored functionally (1 mM L-arginine-induced relaxation), biochemically (appearance of iNOS mRNA), and immunohistochemically. Functional iNOS (L-arginine-mediated relaxation) was induced in RA and CM tissues (but NOT in the LM preparation) over 2 to 5 h. iNOS induction was detected by immunocytochemistry in RA smooth muscle elements and in macrophage-like cells in CM. Functional iNOS induction correlated with iNOS mRNA induction. In the RA and CM, functional iNOS induction was blocked by both actinomycin D and cycloheximide; actinomycin D also blocked the appearance of iNOS mRNA in both tissues. In contrast, cycloheximide blocked CM (but not RA) iNOS mRNA induction. In CM tissue, functional iNOS induction was not affected by genistein, tyrphostin 47/AG213, or vanadate. But, in the RA, both genistein and tyrphostin 47/AG213 blocked the appearance of functional iNOS; neither inhibitor prevented the appearance of RA iNOS mRNA. Vanadate, in the RA tissue, blocked both the appearance of iNOS mRNA and the induction of functional iNOS. In RA tissue, but not in the CM, inhibitors of NF-kappaB activation blocked the appearance of both functional iNOS and iNOS mRNA. We conclude that in different smooth muscle preparations (aorta versus gastric), there can be a differential induction of iNOS mRNA and "functional" iNOS not only in different cellular elements but also in terms of different signaling pathways.

Animals↗
PubMed1999

Effect of diclofenac sodium and dexamethasone on cultured human Tenon's capsule fibroblasts.

BACKGROUND AND OBJECTIVE: To investigate the effect of diclofenac sodium and dexamethasone on cultured human Tenon's capsule fibroblasts. MATERIALS AND METHODS: Two experiments were conducted. In the first experiment, fibroblasts were treated with either diclofenac sodium or dexamethasone at different concentrations, and the cell growth was quantified by using Coulter counter and hexosaminidase methods at 1, 3, 5, and 7 days after adding the drugs. In the second experiment, the cells were treated with each drug for 24 hours and then the cultures were switched to a drug-free medium. The cell growth was quantified at day 7 after removing the drugs from the medium. RESULTS: In the first experiment, inhibition of fibroblast growth in a dose-dependent manner was observed from days 1 to 7 in the cultures treated with each drug. The inhibitory was more pronounced in the diclofenac treated cultures. The typical spindle-shaped fibroblasts treated with higher concentrations of the drugs became spherical cells. In the second experiment, inhibition was not observed when the cultures were switched to a drug-free medium. The spherical cells recovered to spindle-shaped cells and proliferated as normal cells. CONCLUSION: Our results have shown that diclofenac sodium and dexamethasone can significantly inhibit human Tenon's capsule fibroblast growth in a cell culture model. The inhibitory effect was not observed when the cultures were switched after 24 hours to a drug-free culture medium.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Proteinase-activated receptor 2 (PAR(2)): development of a ligand-binding assay correlating with activation of PAR(2) by PAR(1)- and PAR(2)-derived peptide ligands.

A cloned rat proteinase-activated receptor (PAR)(2)-expressing cell line (KNRK-rPAR(2)) was used to study the structure-activity relationships (elevated intracellular Ca(2+)) for a series of: 1) PAR(1)-derived receptor-activating ligands (PAR(1)-APs) [SFLLR (P5), SFLLR-NH(2) (P5-NH(2)), SFLLRNP (P7), SFLLRNP-NH(2) (P7-NH(2)), and TFLLR-NH(2) (TF-NH(2))] and 2) PAR(2)-derived-activating-peptides (PAR(2)-APs) [SLIGRL-NH(2) (SL-NH(2)), SLIGR-NH(2) (GR-NH(2)), and SLIGKV-NH(2) (KV-NH(2))]. The activities of the PAR-APs were compared with the PAR(2)-AP analog trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH(2) tc-NH(2)), which as a [(3)H]propionyl derivative ([(3)H]propionyl-tc-NH(2)) was used to develop a radioligand-binding assay for PAR(2). The relative potencies of the PAR-APs in the Ca(2+)-signaling assay were tc-NH(2) = SL-NH(2) > KV-NH(2) congruent with P5-NH(2) > GR-NH(2) > P7-NH(2) > P7 > P5 > TF-NH(2). The reverse sequence PAR-APs, LSIGRL-NH(2) (LS-NH(2)), LRGILS-NH(2) (LR-NH(2)), FSLLRY-NH(2) (FSY-NH(2)), and FSLLR-NH(2) (FS-NH(2)), as well as the Xenopus PAR(1)-AP TFRIFD-NH(2), were inactive. The relative biological potencies of the peptides were in accord with their ability to compete for the binding of [(3)H]propionyl-tc-NH(2) (tc-NH(2) = SL-NH(2) > GR-NH(2) congruent with P5-NH(2) > P5) to KNRK-rPAR(2) cells, whereas inactive peptides (FS-NH(2); LR-NH(2)) showed no appreciable binding competition. Our data therefore validate a ligand-binding assay for the use in studies of PAR(2) and indicate that the relative biological potencies of the PAR(1)-APs for activating rat PAR(2) parallel their ability to activate human PAR(1). The relative receptor-binding activities of the PAR-APs, although in general agreement with their relative biological activities, point to differences in the intrinsic receptor-activating activities between the several PAR-APs. The binding assay we have developed should prove of use for the further study of PAR(2)-ligand interactions.

Animals↗
PubMed1999

Proteinase-activated receptor 4 (PAR4): action of PAR4-activating peptides in vascular and gastric tissue and lack of cross-reactivity with PAR1 and PAR2.

We studied the actions of the human and murine proteinase-activated receptor 4 (PAR4) derived receptor-activating peptides (APs), GYPGQV-NH2 (GQV-NH2) and GYPGKF-NH2 (GKF-NH2), (i) to activate-desensitize either PAR1 or PAR2 in cultured cell systems (calcium signalling in PAR1/PAR2-bearing human HEK cells and in rat KNRK cells expressing either rat or human PAR2) and (ii) to affect contractility in rat aorta (RA) and rat gastric longitudinal muscle (LM) preparations in vitro. We found that neither PAR1 nor PAR2 was affected by concentrations of the PAR4-APs (800 microM) that caused both an endothelium-dependent nitric oxide mediated relaxation of preconstricted RA tissue and a contractile response in the LM preparation. The potencies (EC50 values 300 to 400 microM) of GQV-NH2 and GKF-NH2 for causing a relaxant effect were identical and comparable with the potency of GQV-NH2 for causing a contractile effect in the LM. However, the potencies of the PAR4-APs in the RA and LM preparations were 20- to 150-fold lower than the potency of the receptor-selective PAR1-AP, TFLLR-NH2. We conclude that the PAR4-APs do not activate either PAR1 or PAR2, and we suggest that along with PAR1 and PAR2, PAR4 may also be present in rat vascular and gastric smooth muscle.

Animals↗
PubMed1999

cSrc is a major cytosolic tyrosine kinase in vascular tissue.

We are interested in identifying, in vascular tissue, nonreceptor tyrosine kinases that may be responsible for the contractile actions of G-protein-coupled agonists such as angiotensin II. By using a series of chromatographic steps, including ion exchange, hydrophobic, and affinity chromatography, we have isolated a major fraction of tyrosine kinase activity from the cytosolic fraction of porcine aorta tissue. According to (i) its immunologic cross-reactivity with the monoclonal anti-cSrc antibody, m327, and with the N-terminally directed monoclonal cSrc2-17 antibody, (ii) its inhibition by the C-terminal cSrc kinase, CSK, and (iii) its specificity for phosphorylating tyrosine 15 in the cdc2(6-20) peptide kinase substrate, we conclude that the kinase we have isolated represents porcine cSrc. A substantial proportion of the enzyme (>70%) was recovered in the cytoplasmic fraction from aorta tissue. The profile of inhibition of the human and porcine cSrc enzymes by a spectrum of tyrosine kinase inhibitors (PP1 >> AG82 > AG490 approximately/= genistein > AG10) was compared with the profile of inhibition of angiotensin II mediated contraction in a porcine coronary vascular preparation (AG10 >> genistein > or = AG82 > or = AG490; PP1 inactive). The different inhibitory profiles indicated that cSrc does not represent the vascular tyrosine kinase responsible for the contractile actions of angiotensin II. We suggest, nonetheless, that cSrc plays a key role for other actions of angiotensin II in intact vascular tissue, such as the regulation of mitogen-activated protein kinase activity and gene transcription.

Amino Acid Sequence↗