Protease-mediated signalling: new paradigms for cell regulation and drug development.
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Amino Acid Sequence↗
Biomedical subjects
M D Hollenberg
Publications and source records attributed to M D Hollenberg.
Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue.
1. The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and SLIGR-NH2 (PP5-NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2. From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3. In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-1; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4. The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of cross-desensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5. In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 > > PP6-NH2 > or = PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6. The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7. In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8. In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > or = PP6 > PP5-NH2 > TP5-NH2). 9. In the rat aortic preparation, the relaxant actions of the PAR-2-derived peptides were mimicked by trypsin, at concentrations (0.5-1 u ml-1; 1-2 nM) lower than those that can activate the thrombin receptor. 10. The bioassay data obtained with the PAR-2 peptides and with trypsin, along with the molecular cloning/RT-PCR analysis, point to the presence of functional PAR-2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR-2-activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.
Animals↗
Epidermal growth factor receptor binding and biological activity in the ovary of goldfish, Carassius auratus.
The receptor binding and biological activity of epidermal growth factor-urogastrone (EGF) was characterized in the follicle-enclosed goldfish oocyte. The binding of 125I-labeled mouse EGF (mEGF) to goldfish ovarian membrane preparation was peptide specific, saturable, reversible, and dependent on time and tissue concentration. Binding data analysis indicated the presence of a single class of high-affinity binding sites with an estimated equilibrium dissociation constant of 4.4 +/- 1.8 x 10(-10) M. The 125I-mEGF binding was displaced by unlabeled mEGF and by human recombinant transforming growth factor-alpha (hTGF-alpha). Both mEGF and hTGF-alpha were found to stimulate reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown (GVBD). Treatment with mEGF and hTGF-alpha stimulated GVBD from a basal level of 8.5 to approximately 30% with an estimated half-maximal effective dose for EGF of 5.80 +/- 0.82 + 10(-10) and for hTGF-alpha, 1.9 +/- 1.0 x 10(-10) M. Furthermore, treatment with mEGF marginally increased 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP)-induced GVBD without significantly influencing the gonadotropin-induced response. Treatment with either mEGF or hTGF-alpha significantly reduced human chorionic gonadotropin-stimulated testosterone production in a concentration-related manner. These data suggest that members of the EGF family may play a role in the regulation of ovarian function in goldfish.
Animals↗
Identification of classical, novel, and atypical protein kinase C isoenzymes in the bovine parathyroid.
Because phospholipid metabolism leading to the activation of protein kinase C (PKC) may play a key regulatory role in the degradation and secretion of PTH, we examined parathyroid cell fractions for the presence of various PKC isoenzymes. Hydroxylapatite chromatography identified the classical PKCs, alpha and beta, but not gamma in parathyroid cell extracts. Western blot analysis confirmed the presence of PKC alpha and beta in these extracts. Of the so-called novel PKCs, Western blot analysis revealed the presence of only one isoenzyme, novel PKC epsilon in parathyroid cell soluble extracts. Western blot analysis using an antibody to the C-terminus of the atypical isoenzyme, PKC zeta, identified a protein of lower molecular weight in addition to PKC zeta. This lower molecular weight protein presumably represents PKC lambda, which shares a high degree of C-terminal sequence similarity with PKC zeta. These findings suggest the possibility that members of all three groups of the PKC family are present and may play a regulatory role in the bovine parathyroid cell.
Animals↗
Proteinase-activated receptor-2 in rat aorta: structural requirements for agonist activity of receptor-activating peptides.
We measured in rat aorta rings the relaxant activity of a number of peptides derived from the activating sequence (SLIGRL, or PP6) of the proteinase-activated receptor-2 (PAR-2). The relaxant action of PP6-NH2 mimicked the action of low concentrations of trypsin (0.5-1 unit/ml; 1-2 nM), was dependent on an intact endothelium, and was blocked by N-omega-nitro-L-arginine methyl ester but not by N-omega-nitro-D-arginine methyl ester. The relaxant actions of PP6, SLIGRL-NH2 (PP6-NH2), SLIGR (PP5), and SLIGR-NH2 (PP5-NH2) were comparable in magnitude, with relative potencies of PP6-NH2 > or = PP6 > PP5-NH2 > PP5. Peptides lacking either a leucine at position 2 (SAIGRL) or an arginine at position 5 (SLIGAL) exhibited markedly reduced or no relaxant activity; nevertheless, the tetrapeptide LIGR-NH2 exhibited low but detectable intrinsic activity. With the use of reverse-transcriptase/polymerase chain reaction, we documented the presence of PAR-2 mRNA in aorta tissue and determined that the rat aorta amino-terminal receptor-activating sequence was the same as that reported for the murine PAR-2 receptor. We concluded that the rat aorta tissue has a PAR-2 receptor that can be activated by peptides as short as four amino acids; the leucine and arginine at positions 2 and 5, respectively, of the proteolytically revealed PAR-2 receptor-activating sequence play key roles in regulating receptor function.
Amino Acid Sequence↗
Vascular actions of thrombin receptor-derived polypeptides: structure-activity profiles for contractile and relaxant effects in rat aorta.
1. Using endothelium-denuded and intact rat aortic rings, we have determined the contractile and relaxant structure-activity profile for a series of thrombin receptor-derived polypeptides (TRPs) based on the human and rat receptor sequences: SFLLR (P5), SFLLR-NH2 (P5-NH2) SFFLR (Rat P5), SFFLR-NH2 (Rat P5-NH2), SFLLRNP (P7), SFLLRNP-NH2 (P7-NH2), SFFLRNP (Rat P7), SFFLRNP-NH2 (Rat P7-NH2), and SFLLRNPNDKYEPF (P14). 2. A contractile response to thrombin and the TRPs in the endothelium-denuded aortic tissue was minimal or absent in preparations obtained from animal weighing less than 180 g (< 6 weeks of age), but increased with animal size, plateauing in tissues derived from animals weighing between 320 and 420 g (about 9 to 14 weeks of age). In contrast, the contractile responses to KCl and noradrenaline did not differ in the tissues and relaxant responses to the TRPs in endothelium-intact aortic preparations were comparable for tissues obtained from either young (< or = 180 g) or older (> or = 320 g) animals. 3. The contractile response of the endothelium-denuded preparation to thrombin and the TRPs showed marked cross-desensitization: the relaxation response of the intact rings did not desensitize to the TRPs. 4. The relative potencies for the TRPs in the aortic contraction assay were comparable to those for the relaxation assay, but were distinct from the relative potencies we measured previously in a rat gastric longitudinal muscle contraction assay. Further, P5 behaved as a partial agonist in the aortic contraction assay, whereas it had been observed to be a full agonist in the gastric contraction assay. 5. The contractile activity of P5-NH2 in endothelium intact aortic rings was low or absent, but in the presence of the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), the contractions in the intact preparation were equivalent to the response of the endothelium-denuded preparation in the absence of L-NAME.6. The contractile response of the endothelium-denuded aortic preparation to P5-NH2 was inhibited by nifedipine and the kinase C antagonist, chelerythrine, but was resistant to the action of indomethacin,tetrodotoxin and the tyrosine kinase inhibitor, genistein.7 We conclude that the receptor system for the TRPs in the aortic smooth muscle elements, responsible for the contractile response, is similar to the aortic endothelial cell receptor responsible for the relaxation response, but is distinct from the receptor that we have previously characterized in gastric longitudinal smooth muscle, results pointing to the presence of receptor subtypes in the vascular and gastric smooth muscle elements.
Amino Acid Oxidoreductases↗
Contractile actions of thrombin receptor-derived polypeptides in human umbilical and placental vasculature: evidence for distinct receptor systems.
1. We studied the structure-activity profiles of four thrombin receptor-derived polypeptides (TRPs) (P5, SFLLR; P5-NH2, SFLLR-NH2; P7, SFLLRNP; P7-NH2, SFLLRN) in contractile human placental artery (PA), umbilical artery (UA) and umbilical vein (UV) preparations and in a human platelet aggregation assay. 2. The contractile actions of the TRPs in the two arterial preparations were endothelium-independent, whereas in the UV tissue a contractile response was observed only in an endothelium-denuded preparation; no endothelium-mediated relaxation responses were observed in any of the vascular preparations. 3. In the three vascular preparations, the contractile responses required extracellular calcium and were attenuated by the tyrosine kinase inhibitor, genistein. 4. The relative contractile orders of potencies of the TRPs in the three vascular preparations were distinct from each other (PA: P7-NH2 > P7 > P5-NH2 > P5; UA: P7-NH2 > or = P5-NH2 approximately = P7 > > P5; UV: P5-NH2 > > P7-NH2 = P7 > > P5) and these were in turn distinct from the potency order observed in the platelet aggregation assay (P5-NH2 > or = P7-NH2 > P7 > > P5). 5. Despite the markedly dissimilar TRP potency orders in the placental artery and umbilical vein preparations, the cDNA sequences for the thrombin receptor obtained by polymerase chain reaction cloning of cDNA from the two tissue sources were identical. 6. We conclude that the four tissues studied possess functionally distinct thrombin receptor systems that interact in a distinct way with agonist peptides. In view of the identity of the thrombin receptor cDNA in the two tissues displaying the most dissimilar structure-activity profiles, we suggest that in different tissues, differences in post-translational receptor processing or differences in receptor-effector coupling interactions may result in unique thrombin receptor systems that can display distinct structure-activity profiles.
Amino Acid Sequence↗
Detection of functional receptors for the proteinase-activated-receptor-2-activating polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle.
We have studied the actions of the proteinase-activated-receptor-2 (PAR2)-activating polypeptide, SLIGRL-NH2 (SLI-NH2), in rat aorta and in gastric longitudinal muscle preparations. In the phenylephrine-precontracted aorta preparation, SLI-NH2 caused an endothelium-dependent relaxation that mimicked the action of low concentrations (0.5 U/mL) of trypsin and that was blocked by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester. In endothelium-free aorta ring preparation, SLI-NH2 caused neither a relaxation nor a contraction. In the gastric longitudinal muscle preparation, SLI-NH2 caused a transient contraction that mimicked the action of trypsin (0.5 U/mL) and that was sensitive to inhibitors of cyclooxygenase (indomethacin) and tyrosine kinase (genistein). Further, using a reverse-transcriptase - polymerase chain reaction (RT-PCR) approach we detected, in both assay tissues, mRNA for the rat PAR2 receptor, and we ascertained, using a cloned receptor cDNA obtained from a rat intestinal cDNA library, that the putative N-terminal activating peptide sequence of the rat PAR2 receptor (SLIGRL) is identical with the one previously cloned from murine tissue. We concluded that, like the thrombin receptor, the PAR2 receptor may play a pathophysiologic role in the regulation of vascular and gastric smooth muscle contractility.
Animals↗
Detection of sarcoma virus family tyrosine kinase activity in coronary arterial tissue.
We have observed that the tyrosine kinase inhibitors genistein and tyrphostin can selectively block angiotensin II mediated and vasopressin-mediated contractions in porcine coronary arterial strips, without affecting the action of acetylcholine. Therefore, we assessed the presence of tyrosine kinase activity in the porcine coronary artery tissue, using an assay specific for sarcoma virus (src) related tyrosine kinases. In both membrane and cytosolic fractions of porcine coronary artery, we detected src-related tyrosine kinase activity that could be inhibited by both genistein and tyrphostin. The tyrosine kinase activity in membrane extracts was separated into two peaks by sequential chromatography on hydroxylapatite and Mono-Q columns. Protein in both peaks exhibited Western blot cross-reactivity with anti-src antibodies and contained tyrosine kinase activity that was inhibited by genistein and tyrphostin. We conclude that porcine coronary artery tissue contains src-related tyrosine kinase activity. However, because of the comparatively low sensitivity of the isolated src kinase activity towards genistein and tyrphostin, compared with the much higher sensitivity of the contractile response to these inhibitors, a direct role for c-rsc in the regulation of contractions elicited by agonists such as angiotensin II and arginine vasopressin cannot yet be assigned.
Amino Acid Sequence↗
Contractile actions of thrombin receptor-derived polypeptides in rat and guinea pig lung parenchymal smooth muscle.
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Amino Acid Sequence↗
Inhibition of GTP-utilizing enzymes by tyrphostins.
Tyrphostins are a group of organic compounds which are widely used as a tool to specifically inhibit protein tyrosine kinases (Yaish, P., Gazit, A., Gilon, C., and levitzki A. (1988) Science 242, 933-935; Gazit, A., Yaish, P., Gilon, C., and Levitzki A. (1989) J. Med. Chem. 32, 2344-2352; Lyall, R. M., Zilberstein, A., Gazit, A., Gilon, C., Levitzki, A., and Schlessinger J. (1989) J. Biol. Chem. 264, 14503-14509; Osherov, N., Gazit, A., Gilon, C., and Levitzki, A. (1993) J. Biol. Chem. 268, 11134-11142). We report here that members of the tyrphostin family inhibit the GTPase activity of transducin and the enzymatic activities of other GTP-utilizing proteins in retinal rod outer segments, such as guanylyl cyclase or fructose-6-phosphate kinase. In contrast, ATP-utilizing enzymes such as hexokinase or rhodopsin kinase were not effected.
Adenosine Triphosphatases↗
Contractile actions of endothelins in rat gastric body: evidence for receptor subtypes and involvement of prostaglandin E2.
Endothelin-1 produced a phasic contraction in the longitudinal muscle preparation isolated from the rat gastric body, but produced a sustained contraction in the circular muscle preparation. Indomethacin, a cyclo-oxygenase inhibitor, decreased the endothelin-1-induced contraction of the longitudinal preparation, but did not affect the endothelin-1-induced contractions of the circular muscle. In the absence of indomethacin, the maximal contractile tension (Emax) and the concentration producing a half-maximal contraction (EC50) induced by endothelin-3 in the longitudinal muscle preparations were smaller than those for endothelin-1, whereas in the circular muscle preparations there were no significant differences between the values (EC50, Emax) for endothelin-1 and endothelin-3. In the presence of indomethacin, endothelin-3-induced contraction of the longitudinal muscle preparation is more potent than that of endothelin-1. SC-19220, a prostaglandin E2 receptor antagonist, significantly decreased endothelin-1-induced contraction of the longitudinal preparation, prostaglandin E2 produced a concentration-dependent contraction in the longitudinal preparation, but had no effects in the circular muscle preparation. Endothelin-1 (10(-8) M) significantly increased the release of immunoassayable prostaglandin E2 from rat gastric smooth muscle. These results point to the existence of distinct endothelin receptor subtypes in the smooth muscle of rat gastric body, and a potential role of endothelin-1 in regulating gastric motility. Moreover, one of the endothelin receptor subtypes is related to the production of prostaglandin E2.
Animals↗
The acute actions of growth factors in smooth muscle systems.
Work over the past six to eight years has established that mitogenic polypeptides such as epidermal growth factor-urogastrone (EGF-URO) or platelet-derived growth factor (PDGF), commonly referred to as 'growth factors', can have rapid (seconds to minutes) regulatory actions in a variety of smooth muscle systems. With EGF-URO as a prototype example, this article describes three distinct smooth muscle response paradigms, two of which (type A and type B) comprise a rapid (seconds to minutes) increase in muscle tension) and one of which (type C) is characterized by an EGF-URO-mediated reduction in sensitivity to other agonists. The quite distinct signal transduction pathways for the three types of response paradigms are outlined, and the marked tissue and species variation in the types of smooth muscle responses that can be observed, even for a single growth factor agonist, are summarized. The article also illustrates that G-protein-coupled vasoactive agents such as angiotensin-II and vasopressin, which can act as 'growth factors' in cultured cell systems, can also work via tyrosine kinase pathways to cause contraction in some of the same intact smooth muscle preparations that contract in response to growth factors such as EGF-URO. Attention is drawn to the fact that many so-called 'growth factors', quite apart from stimulating cell division and differentiation, may in many instances act as rapid localized paracrine/autocrine regulators of tissues such as smooth muscle. It is also pointed out that some of the tyrosine kinase-modulated signal pathways usually associated with the mitogenic action of 'growth factors' may be involved not only in the rapid effects of agents such as EGF-URO in smooth muscle but also in the contractile actions of G-protein-linked agonists.
Animals↗
Tyrosine kinase pathways and the regulation of smooth muscle contractility.
Epidermal growth factor (EGF) mediates three tyrosine kinase-dependent smooth muscle response paradigms, two of which comprise a rapid increase in muscle tension and one of which is characterized by an agonist-mediated reduction in sensitivity to other agents. The three types of response are mediated via distinct signal transduction pathways, and marked tissue and species variation have been observed, even for a single growth factor agonist. Vasoactive agents, such as angiotensin II and vasopressin, that act via G protein-coupled receptors can also work via tyrosine kinase pathways to cause contraction in some of the same intact smooth muscle preparations that contract in response to EGF. In this review, Morley Hollenberg discusses the tyrosine kinase-modulated signal transduction pathways for EGF and also agonists that act via G protein-coupled receptors, and hypothesizes that there may be an intermediary non-receptor tyrosine kinase that may serve as a point of convergence for the contractile actions of these agents in smooth muscle.
Angiotensin II↗
Regulation of vascular and gastric smooth muscle contractility by pervanadate.
1. The contractile actions of vanadate (VO4) and pervanadate (PV, peroxide(s) of vanadate) were studied in rat gastric longitudinal muscle strips and in aortic rings. The roles of extracellular sodium and calcium were evaluated and the potential effects of nerve-released agonists were considered. The possibility that these responses were due to the potentiation of tyrosine kinase activity, as a result of PV-mediated tyrosine phosphatase inhibition was explored with the use of tyrosine kinase inhibitors (genistein, tyrphostin) and by Western blot analysis of phosphotyrosyl proteins in PV-treated tissues. The ability of PV to mimic the action of the tyrosine kinase receptor-associated agonist, epidermal growth factor-urogastrone (EGF-Uro), in the gastric preparation was also studied. 2. PV caused concentration-dependent contractions in both gastric and aorta-derived tissues, with a potency that was 1 to 2 orders of magnitude greater than that of VO4. 3. Although repeated exposure of gastric and aortic tissues to a fixed concentration of VO4 caused reproducible contractions in both tissues, repeated exposure of gastric tissue to PV caused an increased contractile response plateauing after 3 exposures. In contrast, a single exposure of aortic tissue to PV (20 microM) caused a prolonged desensitization of the tissue to the subsequent contractile actions of PV or other agonists. 4. The contractile responses to PV were unaffected in both preparations by tetrodotoxin, atropine, yohimbine and phenoxybenzamine; and in the aortic preparation, the responses to VO4 and PV were the same in the presence or absence of a functional endothelium. 5. PV-induced contractions in both tissues were observed in the absence of extracellular sodium but required extracellular calcium and were attenuated by 1 micro M nifedipine.6. In the gastric preparation, the characteristics of the contractile actions of PV paralleled those of EGF-Uro in terms of (1) inhibition by genistein, (2) inhibition by indomethacin and (3) a requirement for extracellular calcium. These response characteristics differed from those of other contractile agonists such as carbachol.7. In both the gastric and aortic preparations genistein was able to inhibit PV-induced contractions selectively without causing comparable inhibition of KCI-induced contractions. Tyrphostin (AG18) also selectively blocked PV-induced contractions in the gastric, but not in the aortic preparation.8. In both the gastric and aortic tissue, in step with an increased contractile response, PV caused increases in tissue phosphotyrosyl protein content, as detected by Western blot analysis using a monoclonal antiphosphotyrosine antibody; the increases in phosphotyrosyl protein content were reduced when tissues were treated with PV at the same time as a tyrosine kinase inhibitor.9 PV, at sub-contractile concentrations, potentiated the contractile action of angiotensin II in both the gastric and aorta tissue.10 We conclude that the growth factor-mimetic agent, PV, is a much more potent contractile agonist than V04 in both vascular and gastric smooth muscle tissue. PV can cause enhanced tissue phosphotyrosyl protein content most likely via the inhibition of tissue protein tyrosine phosphatases. The contractile actions of PV, which require extracelullar calcium and are independent of extracellular sodium, would appear not to be due either to Na+/Ca2" exchange, promoted by Na+/K+-ATPase inhibition or to the inhibition of Ca2+-ATPase and might be best explained by the ability of PV, via tyrosine phosphatase inhibition, to potentiate a tyrosine kinase pathway linked to calcium entry and to the contractile process.
Angiotensin II↗
Tyrosine kinase inhibitors and the contractile action of G-protein-linked vascular agonists.
In a porcine coronary artery helical strip preparation, the tyrosine kinase inhibitors genistein and tyrphostin (AG82) attenuated the contractile actions of angiotensin II, arginine vasopressin, epidermal growth factor--urogastrone, noradrenaline, and prostaglandin F2 alpha, under conditions where contractions due to acetylcholine and KCl were not affected. Both genistein and tyrphostin also caused a selective inhibition of angiotensin II action in rat aorta helical strips, without affecting KCl-mediated contractions. The IC50 values for the inhibition of contraction in the porcine coronary artery were in the range of 2-5 microM for genistein and 8-15 microM for tyrphostin. Comparable IC50 values were observed for the inhibitory effects of genistein on angiotensin II and prostaglandin F2 alpha action in the rat aorta, whereas much higher tyrphostin concentrations (IC50 > or = 40 microM) were required to block angiotensin II action in this preparation. Angiotensin II caused an elevation of phosphotyrosyl protein (antiphosphotyrosine Western blot) in the porcine coronary artery, which was reversed by genistein. In addition, porcine coronary artery derived membrane and cytosolic fractions exhibited sarcoma virus related tyrosine kinase activity, which was inhibited by both genistein and tyrphostin. Our data (i) document the selective inhibition by genistein and tyrphostin of the contractile action of some, but by no means all, G-protein-linked vascular agonists in porcine and rat arterial preparations, (ii) establish the presence of sarcoma virus related tyrosine kinase activity in the porcine coronary artery, and (iii) demonstrate angiotensin II mediated increases in phosphotyrosyl protein content in porcine coronary artery tissue. These data support the hypothesis that selected G-protein-linked contractile vascular agonists may act in part via the stimulation of nonreceptor tyrosine kinases. The data also indicate the complex actions of the tyrosine kinase inhibitors, even for the same agonist acting in vascular preparations obtained from different species.
Animals↗
Bi-directional control of smooth muscle tension: regulation by tyrosine kinase and tyrosine phosphatase.
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Animals↗
Ligand binding characterization and molecular analysis of distinct epidermal growth factor-urogastrone receptors in cultured smooth muscle and epithelial cells from guinea pig intestine.
In parallel, we measured the receptor binding affinities for epidermal growth factor-urogastrone (EGF-URO) and transforming growth factor-alpha (TGF-alpha) in cultured smooth muscle (GCM) and epithelial (GPC) cells derived from guinea pig intestine. The relative order of binding affinities in the GCM cells was TGF-alpha > EGF-URO, in keeping with the relative order of biological potencies of these polypeptides in a guinea pig gastric circular muscle contractile bioassay. These data established by ligand binding criteria the presence of a TGF-alpha-preferring receptor in the guinea pig. In contrast, there was a reversed order of binding affinities (EGF-URO > TGF-alpha) for the polypeptides in GPC cells, in accord with an identical order of bioassay potencies previously observed in a guinea pig gastric longitudinal muscle contractile bioassay. Using a reverse transcription-polymerase chain reaction approach, we also cloned and sequenced putative EGF-URO receptor ligand binding domain III from each cell type. Although the binding specificity for TGF-alpha and EGF-URO differed in the GCM and GPC cells, the amino acid sequences of receptor domain III were identical in the two cell types. We conclude that the previously measured differences in biological potencies of EGF-URO and TGF-alpha in the contractile bioassay preparations are due to the distinct receptor binding affinities of EGF-URO and TGF-alpha that can be detected in different tissues. However, our data document that the distinct relative binding affinities for EGF-URO and TGF-alpha that can be observed in different cell types from the same species cannot be accounted for solely by the sequence of putative receptor ligand binding domain III.
Animals↗