Bradykinin- and substance P-induced edema formation in the hamster cheek pouch is tyrosine kinase dependent.

The purpose of this study was to determine whether protein tyrosine kinase, a ubiquitous family of intracellular signaling enzymes that regulates endothelial cell function, modulates bradykinin- and substance P-induced increase in macromolecular efflux from the intact hamster cheek pouch microcirculation. Using intravital microscopy, I found that suffusion of bradykinin or substance P (each, 0.5 and 1.0 microM) onto the cheek pouch elicited significant, concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-dextran (FITC-dextran; molecular mass, 70 kDa; P < 0.05). These responses were significantly attenuated by suffusion of genistein (1.0 microM) or tyrphostin 25 (10 microM), two structurally unrelated, nonspecific protein tyrosine kinase inhibitors (P < 0.05). Conceivably, the kinase(s) involved in this process could be agonist specific because genistein was more effective than tyrphostin 25 in attenuating bradykinin-induced responses while the opposite was observed with substance P. Both inhibitors had no significant effects on adenosine (0.5 M)-induced responses (P > 0.5). Collectively, these data suggest that the protein tyrosine kinase metabolic pathway modulates, in part, the edemagenic effects of bradykinin and substance P in the intact hamster cheek pouch microcirculation in a specific fashion.     

PGH(2)-TxA(2)-receptor blockade restores vasoreactivity in a new rodent model of genetic hypertension.

The purpose of this study was to determine whether activation of prostaglandin H(2)-thromboxane A(2) (PGH(2)-TxA(2)) receptors impedes vasodilation in the in situ peripheral microcirculation of spontaneously hypertensive hamsters, a new rodent model of high-renin genetic hypertension. Using intravital microscopy, we found that vasodilation elicited by suffusion of acetylcholine and vasoactive intestinal peptide (VIP), two neurotransmitters localized in perivascular nerves in the peripheral circulation, on the in situ cheek pouch was significantly attenuated in spontaneously hypertensive hamsters relative to age- and genetically matched normotensive hamsters (P < 0.05). However, nitroglycerin-induced vasodilation was similar in both groups. Pretreatment with SQ-29548, a selective and potent PGH(2)-TxA(2)-receptor antagonist, restored acetylcholine- and VIP-induced vasodilation in spontaneously hypertensive hamsters. SQ-29548 had no significant effects on resting arteriolar diameter and on nitroglycerin-induced vasodilation in both groups. SQ-29548 slightly but significantly potentiated VIP- but not acetylcholine-induced vasodilation in normotensive hamsters. Collectively, these data indicate that activation of PGH(2)-TxA(2) receptors impedes agonist-induced vasodilation in the in situ cheek pouch of spontaneously hypertensive hamsters. We suggest that this model is suitable for studying the role of prostanoids in mediating vasomotor dysfunction observed in genetic hypertension.     

Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation

Vascular endothelial growth factor (VEGF) induces mild vasodilation and strong increases in microvascular permeability. Using intravital microscopy and digital integrated optical intensity image analysis, we tested, in the hamster cheek pouch microcirculation, the hypothesis that differential signaling pathways in arterioles and venules represent an in vivo regulatory mechanism in the control of vascular diameter and permeability. The experimental design involved blocking specific signaling molecules and simultaneously assessing VEGF-induced changes in arteriolar diameter and microvascular transport of FITC-Dextran 150. Inhibition of Akt [indirectly via phosphatidylinositol 3-kinase with LY-294002 or wortmannin] or PKC (with bisindolylmaleimide) reduced VEGF-induced hyperpermeability. However, phosphatidylinositol 3-kinase/Akt inhibition enhanced the early phase and attenuated the late phase of VEGF-induced vasodilation, whereas blocking PKC had no effect. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 (with PD-98059 or AG-126) also reduced VEGF-induced hyperpermeability but did not block VEGF-induced vasodilation. Blockade of endothelial nitric oxide synthase (with N(omega)-monomethyl-l-arginine) inhibited VEGF-induced changes in both permeability and diameter. Furthermore, immunofluorescence studies with human umbilical vein endothelial cells revealed that bisindolylmaleimide, PD-98059, and l-NMMA attenuate VEGF-induced reorganization of vascular endothelial cadherin. Our data demonstrate that 1) endothelial nitric oxide synthase is a common convergence pathway for VEGF-induced changes in arteriolar diameter and microvascular permeability; 2) PKC and ERK-1/2 do not play a major role in VEGF-induced vasodilation in the hamster cheek pouch microcirculation; and 3) Akt, PKC, and ERK-1/2 are elements of the signaling cascade that regulates VEGF-stimulated microvascular hyperpermeability. Our data provide evidence for differential signaling as a regulatory step in VEGF-stimulated microvascular dynamics.     

Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells.

Selective inhibitors of tyrosine kinases, tyrphostin 23 and genistein, produced concentration-dependent inhibition of voltage-operated calcium channel currents in vascular smooth muscle cells isolated from rabbit ear artery. The potency of these two structurally dissimilar inhibitors was similar to that reported for their action as inhibitors of tyrosine kinases. Daidzein, an inactive analogue of genistein, had little inhibitory effect on calcium channel currents at concentrations below 300 microM consistent with an action of these agents at a tyrosine kinase. However, tyrphostin 1, a reportedly less active tyrphostin derivative, also inhibited calcium channel currents with a potency similar to tyrphostin 23. These findings suggest that voltage-operated calcium channels in vascular smooth muscle may be modulated by endogenous tyrosine kinase(s) which display different sensitivities to inhibitors compared with the epidermal growth factor (EGF) receptor. Alternatively the possibility of direct blocking actions of these inhibitors at voltage-operated calcium channels cannot be excluded.     

Agonist-induced release of nitric oxide during acute exposure to nicotine

While we have shown that acute infusion of nicotine impairs agonist-induced dilatation of resistance arterioles (Am. J. Physiol. 272:H2337-H2342, 1997), no studies have examined the release of nitric oxide in response to these agonists before and during treatment with nicotine. Thus, the first goal of the present study was to examine agonist-induced release of nitric oxide by the hamster cheek pouch microcirculation under control conditions and during acute infusion of nicotine. We measured the release of nitric oxide (Sievers NO analyzer) in response to repeated topical application of acetylcholine (1.0 microM) and 5'-adenosine diphosphate (ADP; 1.0 microM) during infusion of vehicle and during infusion of nicotine (2.0 microg/kg/min i.v. for 30 minutes followed by a maintenance dose of 0.35 microg/kg/min). In hamsters treated with vehicle, topical application of acetylcholine and ADP elicited reproducible increases in nitric oxide release. In contrast, in hamsters treated with nicotine, there was a marked inhibition of nitric oxide release in response to acetylcholine and ADP. In a previous study (J. Appl. Physiol. 85:1292-1298, 1998) we found that treatment of the hamster cheek pouch microcirculation with superoxide dismutase restored impaired agonist-induced vasodilatation during acute infusion of nicotine. Thus, our second goal was to examine whether superoxide dismutase would restore agonist-induced release of nitric oxide during infusion of nicotine. We found that treatment of the hamster cheek pouch microcirculation with superoxide dismutase prior to infusion of nicotine prevented nicotine-induced impairment of nitric oxide release in response to acetylcholine and ADP. We suggest that nicotine alters dilatation of arterioles via an increased release of superoxide anion and subsequent inactivation of nitric oxide.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

Dexamethasone attenuates grain sorghum dust extract-induced increase in macromolecular efflux in vivo.

The purpose of this study was to determine whether dexamethasone attenuates grain sorghum dust extract-induced increase in macromolecular efflux from the in situ hamster cheek pouch and, if so, whether this response is specific. By using intravital microscopy, we found that an aqueous extract of grain sorghum dust elicited significant, concentration-dependent leaky site formation and increase in clearance of FITC-labeled dextran (FITC-dextran; mol mass, 70 kDa) from the in situ hamster cheek pouch (P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated substance P-induced leaky site formation and increase in clearance of FITC-dextran from the cheek pouch but had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on arteriolar diameter in the cheek pouch. On balance, these data indicate that dexamethasone attenuates grain sorghum dust extract- and substance P-induced increases in macromolecular efflux from the in situ hamster cheek pouch in a specific fashion.     

RPTPmu and protein tyrosine phosphorylation regulate K(+) channel mRNA expression in adult cardiac myocytes

Previously, we reported that cell-cell contact regulates K(+) channel mRNA expression in cultured adult rat cardiac myocytes. Here we show that exposing cardiac myocytes to tyrosine kinase inhibitors (genistein, tyrphostin A25), but not inactive analogs, prevents downregulation of Kv1.5 mRNA and upregulation of Kv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells that endogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells) express the receptor-type protein tyrosine phosphatase mu (RPTPmu) display Kv1.5 mRNA levels paralleling that which was observed in myocytes cultured under high-density conditions and in intact tissue. In contrast, myocytes cocultured with control cells failed to produce this response. Finally, it is shown that Kv4.2 mRNA expression is unaffected by RPTPmu. These findings reveal that multiple tyrosine phosphorylation-dependent mechanisms control cardiac myocyte K(+) channel genes. Furthermore, we conclude that RPTPmu specifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus this receptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart.     

Dexamethasone attenuates acute macromolecular efflux increase evoked by smokeless tobacco extract.

The purpose of this study was to determine whether dexamethasone attenuates the acute increase in macromolecular efflux from the oral mucosa elicited by an aqueous extract of smokeless tobacco (STE) in vivo, and, if so, whether this response is specific. Using intravital microscopy, we found that 20-min suffusion of STE elicited significant, concentration-related leaky site formation and an increase in clearance of fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated the bradykinin-induced leaky site formation and the increase in clearance of FITC-dextran from the cheek pouch. However, it had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on baseline arteriolar diameter and on bradykinin-induced vasodilation in the cheek pouch. Collectively, these data indicate that dexamethasone attenuates, in a specific fashion, the acute increase in macromolecular efflux from the in situ oral mucosa evoked by short-term suffusion of STE. We suggest that corticosteroids mitigate acute oral mucosa inflammation elicited by smokeless tobacco.     

Tyrphostins are inhibitors of guanylyl and adenylyl cyclases

Guanylyl cyclase C (GC-C), the receptor for guanylin, uroguanylin, and the heat-stable enterotoxin, regulates fluid balance in the intestine and extraintestinal tissues. The receptor has an extracellular domain, a single transmembrane spanning domain, and an intracellular domain that harbors a region homologous to protein kinases, followed by the C-terminal guanylyl cyclase domain. Adenine nucleotides can regulate the guanylyl cyclase activity of GC-C by binding to the intracellular kinase homology domain (KHD). In this study, we have tested the effect of several protein kinase inhibitors on GC-C activity and find that the tyrphostins, known to be tyrosine kinase inhibitors, could inhibit GC-C activity in vitro. Tyrphostin A25 (AG82) was the most potent inhibitor with an IC(50) of approximately 15 microM. The mechanism of inhibition was found to be noncompetitive with respect to both the substrate MnGTP and the metal cofactor. Interestingly, the activity of the catalytic domain of GC-C (lacking the KHD) expressed in insect cells was also inhibited by tyrphostin A25 with an IC(50) of approximately 5 microM. As with the full-length receptor, inhibition was found to be noncompetitive with respect to MnGTP. Inhibition was reversible, ruling out a covalent modification of the receptor. Structurally similar proteins such as the soluble guanylyl cyclase and the adenylyl cyclases were also inhibited by tyrphostin A25. Evaluation of a number of tyrphostins allowed us to identify the requirement of two vicinal hydroxyl groups in the tyrphostin for effective inhibition of cyclase activity. Therefore, our studies are the first to report that nucleotide cyclases are inhibited by tyrphostins and suggest that novel inhibitors based on the tyrphostin scaffold can be developed, which could aid in a greater understanding of nucleotide cyclase structure and function.     

Involvement of protein tyrosine kinases and phosphatases in uptake and intracellular replication of virulent and avirulent Leishmania donovani promastigotes in mouse macrophage cells

In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related to the ultimate virulence mechanism associated with intracellular replication of this pathogen. These data collectively suggest that protein tyrosine kinase mediated entry of L. donovani promastigotes into macrophages is not a virulence-associated event.     

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.     

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.     

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.     

Tyrosine kinase inhibitors and the contractile action of epidermal growth factor-urogastrone and other agonists in gastric smooth muscle.

We examined the effects of the tyrosine kinase (TK) inhibitors, genistein, and tyrphostin (RG-50864) on the contractile action of epidermal growth factor - urogastrone (EGF-URO), transforming growth factor-alpha (TGF-alpha), and other agonists in two smooth muscle bioassay systems (guinea pig gastric longitudinal muscle, LM, and circular muscle, CM). We also studied the inhibition by tyrphostin of EGF-URO stimulated protein phosphorylation in identical smooth muscle strips. The selective inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha induced contraction, but not of carbachol- and bradykinin-mediated contraction, occurred at much lower concentrations (genistein, less than 7.4 microM (2 micrograms/mL); tyrphostin, less than 20 microM (4 micrograms/mL)) than those used in previously published studies with these TK inhibitors. In LM tissue, the IC50 values were for genistein 1.1 +/- 0.1 microM (0.30 micrograms/mL; mean +/- SEM) and 3.6 +/- 0.5 microM (0.74 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS: TP) of 1:3 in the longitudinal preparation. In CM tissue, the IC50 values were 3.0 +/- 0.3 microM (0.81 micrograms/mL) for genistein and 2.4 +/- 0.2 microM (0.49 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1.0:0.8 in the circular strips. The inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha mediated contraction was rapid (beginning within minutes) and was reversible upon washing the preparations free from the enzyme inhibitors. In intact tissue strips studied under bioassay conditions, tyrphostin (40 microM) also blocked EGF-URO triggered phosphorylation of substrates detected on Western blots using monoclonal antiphosphotyrosine antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells.

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume-activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 microM) and nifedipine (100 microM) inhibited the hypoosmotically-induced [Ca2+]i increase by more than 90%, while the volume-activated taurine efflux was inhibited by 61.3 +/- 9.5, 74.1 +/- 9.3 and 38.0 +/- 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 microM) and trifluoperazine (10 microM) and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (2 microM) significantly blocked the volume-activated taurine efflux by 93.4 +/- 2.7, 77.9 +/- 3.5 and 61.3 +/- 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12-myristate 13-acetate (100 nM) did not have significant effects on the volume-activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume-activated taurine efflux. These results suggest that the volume-activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.     

Hog barn dust extract increases macromolecular efflux from the hamster cheek pouch.

The purpose of this study was to determine whether short-term exposure to an aqueous extract of hog barn dust increases macromolecular efflux from the intact hamster cheek pouch and, if so, to begin to determine the mechanism(s) underlying this response. By using intravital microscopy, we found that suffusion of hog barn dust extract onto the intact hamster cheek pouch for 60 min elicited a significant, concentration-dependent leaky site formation and increase in clearance of FITC-labeled dextran (molecular mass, 70 kDa). This response was significantly attenuated by suffusion of catalase (60 U/ml), but not by heat-inactivated catalase, and by pretreatment with dexamethasone (10 mg/kg iv) (P < 0.05). Catalase had no significant effects on adenosine-induced increase in macromolecular efflux from the cheek pouch. Suffusion of hog barn dust extract had no significant effects on arteriolar diameter in the cheek pouch. Taken together, these data indicate that hog barn dust extract increases macromolecular efflux from the in situ hamster cheek pouch, in part, through local elaboration of reactive oxygen species that are inactivated by catalase. This response is specific and attenuated by corticosteroids. We suggest that plasma exudation plays an important role in the genesis of upper airway dysfunction evoked by short-term exposure to hog barn dust.     

Tyrosine phosphorylation modulates arteriolar tone but is not fundamental to myogenic response

The present study investigated the role of protein tyrosine phosphorylation in myogenic responsiveness of rat skeletal muscle arterioles. Arteriolar segments were cannulated and pressurized without intraluminal flow. All vessels studied developed spontaneous tone and demonstrated significant myogenic constriction to step changes in pressure with a resultant increase in myogenic tone over an intraluminal pressure range of 50-150 mmHg. Step increases in intraluminal pressure from 50 to 120 mmHg caused a rapid and sustained elevation in intracellular [Ca(2+)], as measured using fura 2. Vessels with myogenic tone dilated in response to tyrosine kinase inhibitors genistein (10 or 30 microM) and tyrphostin A47 (10 or 30 microM) and constricted to the tyrosine phosphatase inhibitor pervanadate (1 or 10 microM). Despite the dilator effect, myogenic reactivity was not blocked by the inhibitors. Daidzein (10 microM), a compound structurally similar to genistein but without tyrosine kinase-inhibiting activity, did not alter vessel tone or myogenic responses. Preincubation of arterioles with genistein or tyrphostin A47 did not significantly alter baseline arteriolar [Ca(2+)], and neither drug reduced the increase in [Ca(2+)] following an acute increase in intraluminal pressure. Constriction induced by pervanadate (10 microM) was not accompanied by a significant increase in intracellular [Ca(2+)], even though removal of extracellular Ca(2+) reversed the constriction. Examination of smooth muscle tyrosine phosphorylation, using a fluorescent phosphotyrosine antibody and confocal microscopy, showed that increased intraluminal pressure resulted in an increase in anti-phosphotyrosine fluorescence. Because manipulation of tyrosine kinase activity was found to alter vessel diameter, these data support a role for tyrosine phosphorylation in modulation of arteriolar tone. However, the results indicate that acute arteriolar myogenic constriction does not require tyrosine phosphorylation.     

Effect of genistein, tyrphostin and herbimycin on prolactin-stimulated progesterone production by porcine theca and luteal cells.

The potential involvement of protein tyrosine kinases (PTK) in the mechanism of prolactin (Prl) action on ovarian cell steroidogenesis has not been elucidated and information about research on this subject is scarce. In this preliminary study pharmacological intervention was used to provide support for a possible involvement of tyrosine kinases in prolactin induction of progesterone secretion by porcine thecal and luteal cells. Material used in this experiment were cultures of porcine follicular theca interna and early corpus luteum cells. The former were isolated from, proestrous preovulatory follicles and the latter were obtained by enzymatic dispersion of luteal tissue. Three of tyrosine kinase inhibitors, genistein, herbimycin and tyrphostin, were applied. They act through different mechanisms, partially blocking Prl-stimulated progesterone secretion. Herbimycin at a dose of 3 microM inhibited Prl-stimulated progesterone secretion beneath the control level in theca and by 70% in luteal cells. Genistein at a dose of 45 microM inhibited Prl-stimulated progesterone secretion beneath the control level in theca and down to the control in luteal cells. On the other hand, tyrphostin at a dose of 100 microM only slightly suppressed Prl-stimulated progesterone secretion by thecal and luteal cells (33% and 40% respectively). This investigation is the first search for evidence of involvement of tyrosine kinases in Prl-stimulated progesterone production by ovarian cells in the pig.     

Conformation and vasoreactivity of VIP in phospholipids: effects of calmodulin

The purpose of this study was to determine the conformation and vasorelaxant effects of vasoactive intestinal peptide (VIP) self-associated with sterically stabilized phospholipid micelles (SSM) and whether calmodulin modulates both of these processes. Circular dichroism spectroscopy revealed that VIP is unordered in aqueous solution at room temperature but assumes appreciable a helix conformation in SSM. This conformational transition was amplified at 37 degrees C and by a low concentration of calmodulin (0.1 nM). Suffusion of VIP in SSM elicited significant time- and concentration-dependent potentiation of vasodilation relative to that elicited by aqueous VIP in the in situ hamster cheek pouch (P < 0.05). This response was significantly potentiated by calmodulin (0.1 nM). Collectively, these data indicate that exogenous calmodulin interacts with VIP in SSM to elicit conformational transition of VIP molecule from a predominantly random coil in aqueous environment to alpha helix in SSM. This process is associated with potentiation and prolongation of VIP-induced vasodilation in the in situ peripheral microcirculation.