Sara F. Rabito

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

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

Captopril improves neurologic outcome from incomplete cerebral ischemia in rats.

We investigated the effects of the angiotensin-converting enzyme inhibitor captopril on neurologic outcome in a rat model of incomplete cerebral ischemia. Twenty male Sprague-Dawley rats were anesthetized with 70% nitrous oxide in oxygen and fentanyl (10 micrograms x kg-1 i.v. bolus, 25 micrograms x kg-1 x hr-1 i.v. continuous infusion). Animals in group 1 (n = 10) received no angiotensin-converting enzyme inhibitor while animals in group 2 (n = 10) were given 10 mg x kg-1 i.v. captopril 30 minutes prior to the ischemic period. Ischemia was produced by unilateral carotid artery ligation and hemorrhagic hypotension to 35 mm Hg for 30 minutes. Body temperature, arterial blood gases, and arterial pH were maintained constant. Neurologic outcome was evaluated every 24 hours for 3 days using a graded deficit score (0, normal; 18, stroke-related death). On the third day after ischemia, captopril significantly improved neurologic outcome (median deficit score = 4) compared with controls (median deficit score = 18) (p less than 0.05). These results suggest that reduced angiotensin II levels or increased tissue kinin concentrations may decrease ischemic brain injury.

Importance of sympathetic innervation in the positive inotropic effects of bradykinin and ramiprilat.

Isolated rat left atria or right ventricular strips were electrically stimulated at a constant frequency. The amplitude of twitch contractions, thus elicited, rose as a function of stimulation intensity because of increases in the evoked release of sympathetic catecholamines. Bradykinin had no effect on contractile force in preparations paced at a minimal intensity (threshold). By contrast, bradykinin (1 nmol/L to 1 mumol/L) markedly increased twitch contractile force when the preparations were paced at a high intensity (two to three times threshold). The EC50 for the positive inotropic action of bradykinin averaged 42 nmol/L. Ramiprilat (1 mumol/L), an angiotensin I-converting enzyme/kinase II inhibitor, shifted the EC50 for bradykinin to approximately 2 nmol/L. Ramiprilat (1 mumol/L) per se also produced a modest positive inotropic effect. The effects of bradykinin and/or ramiprilate were inhibited by HOE 140 (300 nmol/L), a bradykinin B2-receptor antagonist. Propranolol (1 mumol/L), a beta-adrenoceptor blocker, abolished the effects of bradykinin. After the destruction of sympathetic nerve endings by use of 6-hydroxydopamine, bradykinin no longer exerted a positive inotropic action. Cocaine (10 micrograms/mL), an inhibitor of catecholamine reuptake, potentiated the effect of bradykinin. Bradykinin did not affect the positive inotropic response to tyramine (10 mumol/L), whereas cocaine blocked it. Furthermore, bradykinin did not modify the dose-response curves for added norepinephrine. omega-Conotoxin (100 nmol/L) inhibited the positive inotropic effect of intensified stimulation and bradykinin potentiation. Bradykinin is suggested to facilitate the evoked release of sympathetic catecholamines and thereby cause a positive inotropic effect.

Bradykinin B2 Receptors on Skeletal Muscle are Coupled to Inositol 1,4,5-Trisphosphate Formation

To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykimn or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin × bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8] bradykinin with IC50 values of 36.5 ± 6 and 118.0 ± 16.0 pmol/1 from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H ]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/1. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 poml/l, low-affinity IC50 = 22.6 nmol/1) in the absence of guanosine 5′-O-(3-thio-trisphosphate) (GTPγS); they shifted to a model of one affinity (8.1 nmol/1) in the presence of GTPγS. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/1 to 1 μmol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/1) from a basal level of 72.8 ± 16 pmol/mg protein to 433 ± 35.5 at the highest (1 μmol/l) concentration. We conclude that bradykinin B2receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.

Lack of potent antinociceptive activity by substance P antagonist CP-96,345 in the rat spinal cord

Substance P (SP) binds to the NK-1 receptor and has been implicated in the transmission of pain as well as in physiological responses such as salivary gland secretion and neurogenic inflammation. Studies in this field have been limited due to the lack of specific antagonists that are not degraded rapidly and are not neurotoxic. However, a recently developed non-peptide SP antagonist, CP-96,345, is specific for the NK-1 receptor. The purpose of this study was to assess the effects of this antagonist on nociception. The tail flick, paw pinch and hot plate tests were used to assess nociception in rats. Following baseline determination of tail skin temperature and analgesiometric tests, the rats received intrathecal injections of various doses of CP-96,345, and pain sensitivity was assessed at several time intervals up to two hours after injection. The ability of CP-96,345 to inhibit SP induced biting and scratching was also assessed. Results from the analgesiometric tests indicated that there were no significant elevations in the latency of tail flick test or the paw pinch thresholds even at 240 micrograms of CP-96,345. The hot plate latency was elevated at the highest dose of antagonist. In addition, there was a significant dose-related elevation in latency on the hot plate test. CP-96,345 also produced a dose-related decrease in tail skin temperature. CP-96,345 did not block SP induced biting and scratching. CP-96,345 and SP were evaluated for their ability to displace 125I-Tyr8-SP from rat spinal cord, brain and submandibular gland membrane fractions. It was found that although the affinity of CP-96,345 was 56 fold lower than that of SP in the brain, the antagonist was nearly as potent as SP in the spinal cord and submandibular gland. The results of this study suggest that, while CP-96,345 binds to the NK-1 receptor in the spinal cord, this receptor is most likely not involved in mediating some types of nociception at the spinal cord level.

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 ileums 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.

Ketamine relaxes airway smooth muscle contracted by endothelin.

Endothelins (ETs) are synthesized not only in vascular endothelial cells but also in airway epithelial cells.Increased ET-1 has been demonstrated in bronchial epithelium of asthmatic patients, and, in severe asthma attacks, ET-1 increases in plasma and bronchoalveolar lavage fluid. In this study, we investigated whether ketamine (KET) relaxes ET-induced tracheal contractions. Female guinea pigs were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into two strips that were mounted in an organ bath filled with Krebsbicarbonate buffer. The response of each strip to 10-7 M carbachol was taken as 100% contraction to which the response to ET was referred. The contribution of the epithelium to the relaxant effect of KET was studied in denuded tracheae or in the presence of 5 x 10-5 M indomethacin. ET-1 (3 x 10-8 M) induced contractions that were 76 +/- 3% of those induced by carbachol. KET reversed the response to ET-1 in a dose-dependent fashion. Similarly, ET-2 (3 x 10-8 M) induced contractions that were 74 +/- 5% of those induced by carbachol, and KET also reversed this response in a dose-dependent manner. In epithelium-denuded strips, ET-1 induced contractions that were 104 +/- 3% of those induced by carbachol, and KET still reversed this response. The tonic phase of the response to ET-1 was equal (100 +/- 6%) to the response to carbachol, and KET did not affect it significantly. In the presence of ryanodine, KET reduced the ET-1-induced contraction from 67 +/- 2% to 36 +/- 3.%, P < 0.01. In the presence of nicardipine, KET also inhibited the ET-1-induced contraction. We conclude that KET relaxes the tracheal smooth muscle contracted by ETs via a mechanism that is independent of the tracheal epithelium. The relaxant effect of KET on the ET-induced contraction of the trachealis muscle is not dependent upon blockade of 1) sarcolemma influx of Ca (2+) through the dihydropyridine Ca2+ channel or 2) the release of intracellular Ca2+ through the ryanodine-sensitive intracellular Ca2+ channel. It is likely that the action of KET relaxing ET-induced tracheal contractions is at some point of the inositol 1,4,5-trisphosphate signaling pathway. (Anesth Analg 1997;84:900-6)

Thromboxane A2 Mediates the Stimulation of Inositol 1,4,5-Trisphosphate Production and Intracellular Calcium Mobilization by Bradykinin in Neonatal Rat Ventricular Cardiomyocytes

Bradykinin is a mediator of the protection of myocardium by angiotensin I-converting enzyme/kininase II inhibitors. We reported that the activation of B2 bradykinin receptors in neonatal rat cardiac myocytes in primary culture was followed by hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (IP3). Here we examine the regulation of IP3 formation stimulated by bradykinin. Activation of myocytes with 1 mu/L bradykinin increased IP3 production from 117 +/- 8.3 to 1011 +/- 48.6 pmol/mg protein. Treatment of the cells with 10 mu/L indomethacin or 1 mu/L dexamethasone partially blocked this bradykinin-induced response. Moreover, either U73122, a phospholipase C inhibitor, or (p-amylcinnamoyl) anthranilic acid, a phospholipase A2 inhibitor, blunted the IP3 response to bradykinin. Because thromboxane A2 stimulates inositol bisphosphate metabolism in guinea pig atria, we also investigated the effect of the thromboxane A2 receptor antagonist BM 13177 (1 mu/L), which strongly attenuated the stimulated IP3 production. Since thromboxane A2 appears to partly mediate the IP3 response to bradykinin, we examined the effect of the stable thromboxane A2 mimetic U46619. Control cultures were stimulated more by U46619 than by bradykinin (1629 +/- 14.5 versus 1011 +/- 48.6 pmol IP3/mg protein). This property of U46619 was selectively antagonized by BM 13177. Inhibition of either phospholipase C or phospholipase A2 blunted the IP3 response to U46619. Short-term (30 minutes) activation of protein kinase C with phorbol 12-myristate 13-acetate (10 pmol/L to 1 mu/L) attenuated the IP3 accumulation in response to bradykinin; the effect of phorbol 12-myristate 13-acetate was reversed with 1 mu/L staurosporine, a protein kinase C inhibitor. Treatment with 1 microgram/mL cholera toxin or pertussis toxin for 4 hours amplified the IP3 response to 10 nmol/L bradykinin from 570 +/- 20.0 to 1150 +/- 51.3 and to 1016.7 +/- 21.9 pmol/mg protein. Bradykinin mobilized 9.4% of intracellular calcium stores in cardiomyocytes as assessed by chlortetracycline-based fluorometry, and this effect of bradykinin was blocked by BM 13177 or the B2 bradykinin receptor blocker Hoe 140 by more than 70%. In functional studies, bradykinin (1 mu/L) increased by 12% the twitch contractile force of neonatal rat ventricular strips paced at threshold intensity, but this was unaffected by BM 13177. In conclusion, in cardiomyocytes, bradykinin enhances IP3 production mostly via phospholipase A2 stimulation and thromboxane A2 formation. This prostanoid in turn stimulates its receptor and activates phospholipase C, which then splits phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. The effect of bradykinin on phospholipase C, via thromboxane A2, is negatively regulated by protein kinase C activation.

The role of the N-methyl-D-aspartic acid receptor in the relaxant effect of ketamine on tracheal smooth muscle.

Ketamine and magnesium (Mg2+), well known bronchodilators, have been used to treat patients with status asthmaticus.Both can block the N-methyl-D-aspartic acid (NMDA) receptor. NMDA receptors exist in the airway, and their activation seems to be linked to the release actions of sensory neuropeptides resulting in increased airway tone. We sought to determine whether ketamine relaxes the guinea pig trachea contracted by histamine by blocking the NMDA receptor. Female guinea pigs (250-400 g) were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into strips 3 mm wide and 15 mm long. The strips were mounted in a 10-mL organ bath filled with Tyrodes solution bubbled through with 95% O2/5% CO2 at 37[degree sign]C. Strip contractions were measured isometrically with a force displacement transducer. We then studied the effect of NMDA receptor antagonists on histamine-induced tracheal contraction. In this protocol, we examined the effect of ketamine, Mg2+, zinc (Zn2+), or MK-801 (a noncompetitive NMDA receptor blocker) on strips contracted by 10-5 M histamine. After full contraction was attained, ketamine (0.5-1.5 mM), MgSO4 (2-8 mM), ZnCl2 (0.2-0.8 mM), or MK-801 (1.5-6 x 10-5 M) was added, and the strip tension was measured again. We also studied the effect of NMDA on the relaxation by ketamine. After full contraction by 10-5 M histamine, 0.5-1.5 mM KET was added alone or in combination with 0.1 mM NMDA, and the strip tension was measured again. Finally, we measured the effect of MK-801 on the relaxant effect of ketamine. After full contraction by 10-5 M histamine, 0.5-2 mM ketamine was added alone or in combination with 0.75 or 1.5 x 10-5 M MK-801, and the strip tension was measured again. All NMDA receptor antagonists tested reversed the tracheal contraction induced by histamine in a dose-dependent manner. However, neither the agonist NMDA nor the noncompetitive receptor blocker MK-801 affected tracheal relaxation induced by ketamine. We conclude that ketamine relaxes the tracheal smooth muscle contracted by histamine through a mechanism independent of NMDA receptors. The decreased bronchomotor tone induced by ketamine is probably due to interference with a Ca2+-requiring step necessary to maintain the contraction caused by histamine. Implications: Stimulation of the N-methyl-D-aspartic acid (NMDA) receptor in the airway results in airway constriction. The bronchodilator ketamine blocks the NMDA receptor. However, ketamine relaxes the guinea pig trachea contracted by histamine through a mechanism independent of the NMDA receptor. (Anesth Analg 1998;87:1383-8)

The Relaxant Effect of Ketamine on Guinea Pig Airway Smooth Muscle Is Epithelium-Independent

Airway epithelial cells and vascular endothelial cells modulate the tone of the underlying smooth muscle by releasing relaxing factors such as prostanoids and nitric oxide (NO).In the present study, we investigated whether the relaxant effect of ketamine depends on any of the epithelium-derived relaxing factors. Tracheae of female guinea pigs were cut spirally into strips (15 x 3 mm) and mounted in water-jacketed organ baths filled with Krebs-bicarbonate buffer aerated with a mixture of 95% O (2) and 5% CO2 at 37 degrees C. Changes in the tension of the strips were measured isometrically with a force displacement transducer and recorded with a polygraph. In the first set of experiments, we examined the effect of ketamine on the concentration-response curves for histamine and carbachol in strips in which the epithelium was kept intact and in strips with denuded epithelium. In the second and third set of experiments, we studied the effect of indomethacin, a cyclooxygenase inhibitor, and N-omega-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor, on the relaxant activity of ketamine on tracheal strips contracted by histamine or carbachol. The following results were obtained: 1.Mechanical denudation of the tracheal epithelium shifted the concentration-response curve for histamine to the left (the 50% effective concentration [EC50] value of histamine decreased from 3.5 +/- 0.02 x 10-6 M in the intact strips to 0.98 +/- 0.01 x 10-6 M in denuded strips, P < 0.001). However, removal of the tracheal epithelium did not change the response to carbachol (the EC50 for carbachol was 1.1 +/- 0.02 x 10-7 M in intact strips versus 0.88 +/- 0.01 x 10-7 M after epithelial removal, P > 0.05). 2.Ketamine shifted to the right the concentration-response curves for histamine and carbachol in both intact and denuded tracheae. 3.Indomethacin did not alter the relaxant effect of ketamine on the tracheae contracted by either histamine (the concentration that inhibits 50% [IC50] of ketamine = 1.5 +/- 0.01 x 10-3 M in control strips and 1.3 +/- 0.04 x 10-3 M in strips pretreated with indomethacin, P > 0.05) or carbachol (the IC50 of ketamine was 2.5 +/- 0.02 x 10-4 M in control strips and 2.4 +/- 0.01 x 10 (-4) M in strips pretreated with indomethacin, P > 0.05). 4.L-NAME did not influence the relaxant effect of ketamine on tracheae contracted by either histamine (the IC50 of ketamine = 1.6 +/- 0.05 x 10-3 M in control strips and 1.6 +/- 0.05 x 10-3 M in strips pretreated with L-NAME, P > 0.05) or carbachol (the IC50 of ketamine = 2.6 +/- 0.04 x 10-4 M in control strips and 2.3 +/- 0.01 x 10-4 M in trips pretreated with L-NAME, P > 0.05). These results indicate that neither the mechanical removal of the tracheal epithelium nor the blockade of the release of potent mediators from tracheal epithelial cells influence the relaxant effect of ketamine on guinea pig tracheal strips contracted by histamine or carbachol.We conclude that ketamine relaxes the airway smooth muscle by an epithelium-independent mechanism. (Anesth Analg 1997;84:641-7)