Potentiation of the effects of bradykinin on its receptor in the isolated guinea pig ileum

Angiotensin I-converting enzyme (ACE/kininase II) inhibitors potentiated guinea pig ileum's isotonic contractions to bradykinin (BK) and its analogues, shifting the BK dose-response curve to the left. ACE inhibitors added at the peak of the contraction immediately enhanced it further (343 +/- 40%), although the ileum inactivated BK slowly (t(1/2) = 12-16 min). Chymotrypsin and cathepsin G also augmented the activity of BK up to three- or four-fold, but in a manner slower than that of ACE inhibitors. The BK B(2) receptor blocker HOE 140 inhibited all effects. Histamine and angiotensin II were not potentiated. ACE inhibitors potentiate BK independent of blocking its inactivation by inducing crosstalk between ACE and the BK B(2) receptor; proteases activate the receptor by different mechanism.     

Monoclonal antibodies to bradykinin inhibit smooth muscle contractile action of bradykinin

Four hybridoma cell lines have been established that secrete monoclonal antibodies to nonapeptide bradykinin. Bradykinin coupled to ovalbumin, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, was used to immunize BALB/c mice. Spleen cells from the immunized animals were fused to P3-X63-AG8-653 mouse myeloma cells. The resultant hybrid cells were screened by enzyme-linked immunoassay for production of antibodies to bradykinin. Hybrids from four positive wells were subcloned by limiting dilution and expanded as ascites tumor into pristane-primed mice. All the four hybrids secreted monoclonal antibodies of IgG1 (k) isotype. Unlabeled peptides bradykinin, lysyl-bradykinin (kallidin) and methionyl-lysyl-bradykinin competed with the radiolabeled [Tyr1]kallidin for monoclonal antibody binding sites. These antibodies recognized preferentially either NH2- or COOH-terminals of the nonapeptide bradykinin and can distinguish between des-Arg1-bradykinin and des-Arg9-bradykinin. Bradykinin fragments smaller than eight residues were not recognized by these antibodies. Monoclonal antibodies BK-D6A5, BK-B6C9 and BK-A3D9 neutralized the smooth muscle contractile activity of bradykinin. An enzyme-linked immunoassay developed using these monoclonal antibodies showed the effective range of bradykinin determination between 5 and 150 ng.     

Staurosporine inhibition of zipper-interacting protein kinase contractile effects in gastrointestinal smooth muscle.

Zipper-interacting protein kinase (ZIPK) is a serine-threonine kinase that has been implicated in Ca2+-independent myosin II phosphorylation and contractile force generation in vascular smooth muscle. However, relatively little is known about the contribution of this kinase to gastrointestinal smooth muscle contraction. The addition of a recombinant version of ZIPK that lacked the leucine zipper domain to permeabilized ileal strips evoked a Ca2+-independent contraction and resulted in myosin regulatory light chain diphosphorylation at Ser19 and Thr18. Neither Ca2+-independent force development nor myosin regulatory light chain phosphorylation was elicited by the addition of kinase-dead ZIPK to the ileal strips. The sensitivity of ZIPK-induced contraction to various kinase inhibitors was similar to the in vitro sensitivity of purified ZIPK to these inhibitors. Staurosporine was the most effective ZIPK inhibitor, with a Ki value calculated to be 2.6 +/- 0.3 micromol/L. Through the use of specific kinase inhibitors, we determined that Rho-associated protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C) do not mitigate ZIPK-induced contraction in ileum. Our findings support a role for ZIPK in Ca2+-independent contractile force generation in gastrointestinal smooth muscle.     

TGF-beta potentiates airway smooth muscle responsiveness to bradykinin

The molecular mechanisms by which bradykinin induces excessive airway obstruction in asthmatics remain unknown. Transforming growth factor (TGF)-beta has been involved in regulating airway inflammation and remodeling in asthma, although it is unknown whether TGF-beta can modulate bradykinin-associated bronchial hyperresponsiveness. To test whether TGF-beta directly modulates airway smooth muscle (ASM) responsiveness to bradykinin, isolated murine tracheal rings were used to assess whether TGF-beta alters ASM contractile responsiveness to bradykinin. Interestingly, we found TGF-beta-treated murine rings (12.5 ng/ml, 18 h) exhibited increased expression of bradykinin 2 (B(2)) receptors and became hyperreactive to bradykinin, as shown by increases in maximal contractile responses and receptor distribution. We investigated the effect of TGF-beta on bradykinin-evoked calcium signals since calcium is a key molecule regulating ASM excitation-contraction coupling. We reported that TGF-beta, in a dose- (0.5-10 ng/ml) and time- (2-24 h) dependent manner, increased mRNA and protein expression of the B(2) receptor in cultured human ASM cells. Maximal B(2) receptor protein expression that colocalized with CD44, a marker of membrane cell surface, occurred after 18 h of TGF-beta treatment and was further confirmed using fluorescence microscopy. TGF-beta (2.5 ng/ml, 18 h) also increased bradykinin-induced intracellular calcium mobilization in fura-2-loaded ASM cells. TGF-beta-mediated enhancement of calcium mobilization was not attenuated with indomethacin, a cyclooxygenase inhibitor. These data demonstrate for the first time that TGF-beta may play a role in mediating airway hyperresponsiveness to bradykinin seen in asthmatics by enhancing ASM contractile responsiveness to bradykinin, possibly as a result of increased B(2) receptor expression and signaling.     

Presence of specific bradykinin receptors in arterial and venous smooth muscle

The relationship between bradykinin action and its concentration was examined on isolated rings of the rabbit aorta, femoral artery, jugular vein and on isolated strips of the rat portal vein. The sensitivity of femoral artery and portal vein smooth muscles to bradykinin was disclosed. Venous smooth muscles were more sensitive to bradykinin as compared with arterial smooth muscles. Dissociation constants for the rabbit aorta, femoral artery, jugular vein and for the rat portal vein were 3.98 X 10(-6), 6.3 X 10(-6), 1.26 X 10(-7), and 7.6 X 10(-9)M, respectively. Effects of endogenous bradykinin in vivo might result from its primary action on the venous smooth muscle, action on the arterial smooth muscle and veno-arterial interactions.     

Potentiation and inhibition of nicotinic effects on striated muscle by the tetramine disulfide benextramine

In low concentrations (0.3-3 muM) the tetramine disulfide benextramine (BHC; N,N'-bis[6-(o-methoxybenzylamino)-n-hexyl]cystamine) potentiated the contracture of the isolated frog rectus abdominis muscle caused by acetylcholine but in the presence of physostigmine or in a higher concentration (10 muM) it inhibited. Benextramine only inhibited the contracture caused by carbachol or butyrylcholine. The all-carbon analog of benextramine only inhibited the effect of acetylcholine. The inhibitory effects of benextramine and its carbon analog were noncompetitive and readily reversible but the potentiating effect of benextramine was not readily reversible.     

Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.     

Ca2+-dependent inhibition of smooth muscle adenylate cyclase activity

We have examined the inhibitory regulation by Ca2+ of the adenylate cyclase activity associated with microsomes isolated from bovine aorta smooth muscle. In the presence of 2 mM MgCl2, Ca2+ (0.8-100 microM) inhibited in a noncompetitive manner activation of the enzyme by GTP, Gpp[NH]p, or forskolin. In all instances the value for half-maximal inhibition was between 2 and 3 microM. In contrast, Ca2+ inhibited the activation by MgCl2 (2-50 mM), alone or in the presence of GTP, in a competitive manner. The inhibition of adenylate cyclase by 10 microM Ca2+ was reversed in the presence of either 5 or 25 microM calmodulin or troponin C. These data show that (i) Ca2+, at concentrations similar to those which activate smooth muscle contraction, inhibits the stimulation of adenylate cyclase by several activators; (ii) Ca2+ and Mg2+ compete for a common site on the smooth muscle adenylate cyclase complex; and (iii) the reversal of Ca2+-dependent inhibition by Ca2+-binding proteins may be produced by chelation of the metal by these proteins.     

Actomyosin isolated from bovine tracheal smooth muscle.

A contractile protein (actomyosin) was isolated from bovine tracheal smooth muscle by the use of "classical" procedures. The protein was considered to be actomyosin because it demonstrated: ATPase activity; superprecipitation upon the addition of ATP, and the solubility and extraction characteristics of actomyosin. The ATPase and superprecipitation reactions were not inhibited by EGTA, and did not require calcium. Lack of an effect of either calcium or EGTA could not be reversed by the addition of active bovine skeletal muscle troponin and tropomyosin. No troponin-tropomyosin like activities could be demonstrated in various tracheal muscle fractions.     

Mechanisms of MAPK activation by bradykinin in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is a prominentfeature of the atherosclerotic process occurring after endothelial injury. A vascular wall kallikrein-kinin system has been described. Thecontribution of this system to vascular disease is undefined. In thepresent study we characterized the signal transduction pathway leadingto mitogen-activated protein kinase (MAPK) activation in response tobradykinin (BK) in VSMC. Addition of10¹⁰-10⁷M BK to VSMC resulted in a rapid and concentration-dependent increasein tyrosine phosphorylation of several 144- to 40-kDa proteins. Thiseffect of BK was abolished by theB₂-kinin receptor antagonistHOE-140, but not by the B₁-kininreceptor antagonist des-Arg⁹-Leu⁸-BK.Immunoprecipitation with anti-phosphotyrosine antibodies followed byimmunoblot revealed that10⁹ M BK induced tyrosinephosphorylation of focal adhesion kinase (p125^FAK). BK(10⁸ M) promoted theassociation of p60^src with theadapter protein growth factor receptor binding protein-2 and alsoinduced a significant increase in MAPK activity. Pertussis and choleratoxins did not inhibit BK-induced MAPK tyrosine phosphorylation. Protein kinase C downregulation by phorbol 12-myristate 13-acetate and/or inhibitors to protein kinase C,p60^src kinase, and MAPK kinaseinhibited BK-induced MAPK tyrosine phosphorylation. These findingsprovide evidence that activation of theB₂-kinin receptor in VSMC leads togeneration of multiple second messengers that converge to activateMAPK. The activation of this crucial kinase by BK provides a strongrationale to investigate the mitogenic actions of BK on VSMCproliferation in disease states of vascular injury.

     

Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase

Systematically synthesized derivatives of ML-9, 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine, were found to inhibit both Ca2+-calmodulin-dependent and -independent smooth muscle myosin light chain kinases with a similar concentration dependence, and their inhibitions were of the competitive type with respect to ATP. Moreover, ML-9 as well as ATP or ADP exhibited an effective protection to inactivation of smooth muscle myosin light chain kinase by the nucleotide affinity label 5'-p-fluorosulfonylbenzoyladenosine, suggesting that ML-9 binds at or near the ATP-binding site on the kinase molecule. These derivatives, which were structurally unrelated to ATP and exhibited more hydrophobic properties detected by reverse-phase high-performance liquid chromatography, exhibited more potent inhibition toward smooth muscle myosin light chain kinase, indicating that the hydrophobic properties of these derivatives positively correlated well with their potencies of inhibiting the catalytic activity for the enzyme. These findings suggest that the ATP-binding site at the active center of smooth muscle myosin light chain kinase is located in a hydrophobic environment. The potent vaso-relaxing effect of ML-9 on rabbit vascular strips and on saponin-treated skinned smooth muscle cells was discussed in relation to the in vivo inhibition by this drug of smooth muscle myosin light chain kinase.     

Effect of mefloquine on the mechanical activity of the mouse isolated rectal smooth muscle.

The effects of mefloquine on the mechanical activity of the mouse isolated rectal smooth muscle was studied. Mefloquine (4.1x10-5 - 5.2x10-3M) when applied alone and separately exerted variable effects on the rectum. In some preparations, it caused slight phasic contractions while in others no response was elicited. When the external Ca(2+) was increased from 1.8mM to 300mM mefloquine produced phasic contractile activity which was abolished on return to normal 1.8mM suggesting that the contractile activity was due to extracellular Ca(2+) influx. Meflaquine [4.1x10-6M - 4.1x10-4M] caused contraction - dependent inhibition of KCL, Carbachol and CaCl2 [in depolarizing Tyrode Solution]. Mefloquine [2.1x10-4M] blocked KCL, but not carbachol contractions which were largely reversed by increasing [Ca2+]. The results show that mefloquine possesses anticholinergic and appreciable calcium channel blocking activity.