Bradykinin Superfusion Research Articles

Norepinephrine reduces heat responses of cutaneous C-fiber nociceptors in Sprague–Dawley rats in vitro

Nociceptors are excited or sensitized by many inflammatory mediators as well as by elevation of tissue temperature. We have shown that there is a facilitatory synergistic interaction between norepinephrine (NE) and bradykinin (BK) on cutaneous C-fiber nociceptors in normal Lewis rats. These interactions may play an important role in the mechanism of sympathetically maintained pain. In the present experiment, using skin-saphenous nerve in vitro preparations, we tested the effect of NE on the activity of nociceptive fibers and their response to heat in normal Sprague–Dawley rats. For comparison with the previous data on Lewis rats, we also examined the effect of NE on BK response. NE (10 −5 or 10 −6 M) did not excite nociceptive fibers before repeated heat stimuli or BK superfusion (10 −5 or 10 −6 M) to the receptive field. In contrast, after a few applications of heat or BK, NE excited 20–43% of nociceptive fibers to similar magnitudes. We also found that NE sensitized subsequent BK responses, but somewhat unexpectedly that it suppressed subsequent heat responses. This occurred regardless of the presence or absence of NE-induced excitation. These results suggest different mechanisms of NE modification to the BK and heat responses of cutaneous C-fiber nociceptors.

Bradykinin-induced proinflammatory signaling mechanisms.

Intravital microscopic techniques were used to examine the mechanisms underlying bradykinin-induced leukocyte/endothelial cell adhesive interactions (LECA) and venular protein leakage (VPL) in single postcapillary venules of the rat mesentery. The effects of bradykinin superfusion to increase LECA and VPL were prevented by coincident topical application of either a bradykinin-B(2) receptor antagonist, a cell-permeant superoxide dismutase (SOD) mimetic or antioxidant, or inhibitors of cytochrome P-450 epoxygenase (CYPE) or protein kinase C (PKC) but not by concomitant treatment with either SOD, a mast cell stabilizer, or inhibitors of nitric oxide synthase, cyclooxygenase, xanthine oxidase, NADPH oxidase, or platelet-activating factor. Immunoneutralizing P-selectin or intercellular adhesion molecule-1 (ICAM-1) completely prevented bradykinin-induced leukocyte adhesion and emigration but did not affect VPL. On the other hand, stabilization of F-actin with phalloidin prevented bradykinin-induced leukocyte emigration and VPL but did not alter leukocyte adhesion. These data indicate that bradykinin induces LECA in rat mesenteric venules via a B(2)-receptor-initiated, CYPE-, oxidant- and PKC-mediated, P-selectin- and ICAM-1-dependent mechanism. Bradykinin also produced VPL, an effect that was initiated by stimulation of B(2) receptors and involved CYPE and PKC activation, oxidant generation, and cytoskeletal reorganization but was independent of leukocyte adherence and emigration.

Sustained sensitization and recruitment of rat cutaneous nociceptors by bradykinin and a novel theory of its excitatory action.

Excitation and sensitization to heat of nociceptors by bradykinin (BK) were examined using an isolated rat skin-saphenous nerve preparation. A total of 52 C-fibres was tested: 42 were mechano-heat sensitive (CMH) and 40% of them were excited and sensitized to heat by BK superfusion (10-5 M, 5 min) of their receptive fields; heat responses were augmented by more than five times and heat thresholds dropped to 36.4 degrees C, on average. Sixty per cent of the CMH did not respond to BK itself, but 3/4 of these units showed an increase in their heat responses by more than 100% following BK exposure. Ten high-threshold mechanosensitive C-fibres did not discharge upon BK application but following this five of them responded to heat in a well-graded manner. In all fibres, the sensitizing effect of BK was abolished within 9 min or less of wash-out, and it could be reproduced several times at equal magnitude, whereas the excitatory effect of BK regularly showed profound tachyphylaxis. Sustained superfusion (20 min) of BK induced a desensitizing excitatory response while superimposed heat responses showed constant degrees of sensitization. The large extent and high prevalence of BK-induced sensitization (almost 80% of all fibres tested) and de novo recruitment of heat sensitivity suggest a prominent role of BK not only in hyperalgesia but also in sustained inflammatory pain which may be driven by body or even lower local temperatures acting on sensitized nociceptors. Based on the latter assumption, a hypothesis is put forward that excludes a direct excitatory effect of BK on nociceptors, but assumes a temperature-controlled activation as a result of rapid and profound sensitization.

Kinin-induced prolongation of action-potential duration in right ventricular muscle from rat: involvement of B1 and B2 receptors.

Previous work has shown that, in rat ventricular muscle, bradykinin (BK) causes a dose-dependent increase in action potential duration (APD), an action that may be responsible for APD prolongation by captopril (kininase II). To determine which kinin receptor might be involved in APD prolongation, we studied the effects of B1- and B2-receptor agonists, as well as those of antagonists and mergepta (a kininase I inhibitor) added during BK superfusion. Action potentials were recorded by using the standard glass microelectrode technique in rat ventricular muscle preparations. Action-potential characteristics were compared between preparations superfused with peptide/drug-free Tyrode's solution (control group) and preparations superfused with peptide/drug-containing solution. APD was significantly longer in preparations superfused with BK (10(-8) M) than in the control group. The APD prolongation induced by BK, a known B2-receptor agonist, was significantly reduced by Hoe 140 (a B2 antagonist) and also by Lys[Leu8]des-Arg9-BK (a B1 antagonist), an action presumably related to inhibition of B1 receptor stimulation by the BK metabolite des-Arg9-BK. When mergepta was added in the presence of BK, APD prolongation by BK was significantly reduced, an effect that could have been related to reduced B1-receptor stimulation after inhibition of the endogenous generation of des-Arg9-BK by kininase I. Sar4-[d-Phe8]des-Arg9-BK, a B1-receptor agonist that is not degraded by kininase II, also prolonged APD. We conclude that both B1 and B2 receptors may be involved in APD prolongation induced in rat ventricular muscle preparations.

Bradykinin and changes in microvascular permeability in the hamster cheek pouch: role of nitric oxide.

1. The objective of this study in the hamster cheek pouch was to investigate the role of nitric oxide in bradykinin-induced microvascular leakage. The cheek pouch microcirculatory bed of the anaesthetized hamster was directly observed under microscope and vascular leakage was evidenced by dextranfluorescein isothiocyanate (FITC-dextran) extravasation. 2. Bradykinin superfusion (but not [des-Arg9]-bradykinin up to 3 x 10(-6) M) induced an increase in microvascular permeability (log EC50: -6.5 +/- 0.4) which was exclusively located on the post-capillary venule. Plasma extravasation was blocked by intravenous pretreatment with Hoe 140, a bradykinin B2 receptor antagonist (estimated log ID50: -9.5 +/- 0.2). 3. The effects of bradykinin (3 x 10(-7) M) superfusion were partially but significantly inhibited by indomethacin (10(-5) M, P < 0.05) and abolished by pretreatment with L-nitro-arginine (L-NOARG; 10(-5) M). 4. Acetylcholine (10(-6) M, which releases endothelial nitric oxide (NO), and sodium nitroprusside (10(-6) M, a nitrovasodilator) superfusion did not induce any changes in permeability, per se. Cromakalim (10(-5) M, a potassium channel opener) superfusion induced a moderate but significant plasma extravasation. 5. The effects of bradykinin, blocked by L-NOARG pretreatment, were restored by the co-perfusion of either sodium nitroprusside or cromakalim. Conversely vasoconstriction, produced by a stable analogue of thromboxane A2 (U46619, 3 x 10(-7) M), inhibited the increase in permeability produced by bradykinin. 6. The measurement of arteriolar diameter showed that bradykinin induced a vasodilatation which was blocked by L-NOARG. L-NOARG in itself was a powerful vasoconstrictor. Sodium nitroprusside and cromakalim, in the presence of L-NOARG, were able to restore the inhibited vasodilator response to bradykinin. 7. These results suggest: (1) bradykinin-induced microvascular leakage is mediated by bradykinin B2 receptor activation; (2) the increase in permeability is due to two different independent phenomena, i.e. post-capillary venular endothelial gap formation and arteriolar vasodilatation which increases the post-capillary venular transmural pressure: (3) NO is only involved in the arteriolar dilatation component of the bradykinin-induced increase in microvascular permeability.

Bradykinin depolarises the rat isolated superior cervical ganglion via B 2 receptor activation

Experiments were undertaken to characterise the action of kinins on sympathetic neurones of the rat superior cervical ganglion (SCG) by use of in vitro greae-gap, extracellular recording techniques in conjunction with selective agonists and antagonists for B 1 and B 2 bradykinin (BK) receptors. Superfusion of BK (10 nM to 10 μM) to the ganglion produced a concentration-related depolarisation (pD 2 = 7.02 ± 0.04, n = 7) which was inhibited by the selective B 2 antagonist HOE 140 (10–100 nM), but not by the B 1 antagonist Leu 8desArg 9 BK (1 μM), indomethacin (7 μM) or the nitric oxide synthase inhibitor l- N G-nitroarginine methyl ester (300 μM). DesArg 9BK (10 nM to 10 μM) had no effect on membrane potential. Pre-treatment of animals with intravenous bacterial lipopolysaccharide (LPS, 3 mg kg −1) failed to induce B 1 receptor-mediated depolarisations of SCG neurones, or change responses to BK ( P > 0.05, n = 4). These experiments highlight and characterise the action of BK as a neuromodulator of sympathetic neurones via B 2 receptor activation.

Cigarette smoke extract potentiates bradykinin-induced increases in microvascular permeability

The first goal of this study was to determine whether cigarette smoke extract (CSE) increases microvascular permeability of the hamster cheek pouch in vivo. The second goal was to determine whether CSE potentiates bradykinin-induced increases in vascular permeability in the hamster cheek pouch. Using intravital microscopy, we examined the permeability of the hamster cheek pouch to fluorescein isothiocyanate-dextran (mol wt 70,000). Increases in permeability were quantitated by counting the number of postcapillary venular leaky sites per 0.11 cm2. Superfusion of CSE (1, 5, and 10%) did not produce venular leaky sites and, thus, did not alter macromolecular permeability. Superfusion of bradykinin (0.1, 0.5, and 1.0 microM) produced a dose-related increase in the number of venular leaky sites. Formation of leaky sites in response to bradykinin was potentiated by CSE. To determine whether potentiation of bradykinin-induced leaky site formation by CSE was related to products released via the cyclooxygenase pathway, we examined the effects of pretreatment with indomethacin (10 mg/kg i.v.). Indomethacin did not alter the potentiating effect of CSE on bradykinin-induced leaky site formation. These findings suggest that CSE does not alter basal permeability of the hamster cheek pouch microcirculation in vivo. However, CSE potentiates bradykinin-induced increases in microvascular permeability. The mechanism of CSE-induced potentiation of microvascular permeability does not appear to be related to substances produced via the cyclooxygenase pathway.

Conditioning of histamine by bradykinin alters responses of rat nociceptor and human itch sensation

The effect of a conditioning bradykinin application on histamine induced excitation of cutaneous nociceptors and on histamine induced sensations of volunteers was studied. Using an in vitro skin nerve preparation, unmyelinated polymodal nociceptor units of rats ( n = 11) were tested by bathing their receptive fields from the corium side with 10 −5 M solutions of bradykinin and histamine. Following bradykinin superfusion the histamine induced discharges were enhanced, and previously unresponsive units were excited by histamine. Corresponding psychophysical experiments were carried out in 13 healthy volunteers. Histamine iontophoresis (30 mC) induced predominantly itching sensations after an intracutaneous control injection of physiological saline. However, following bradykinin injections (100 μl of a 10 −7 M solution) histamine induced little itch but rather a burning sensation lasting 1–2 min. Itching remained suppressed even after the burning sensations had subsided. These data support a hypothesis according to which itching is mediated by a sub-population of polymodal nociceptor units, and pain is induced whenever a larger nociceptor population is recruited. In the CNS itch processing is either occluded (masked) by pain processing, or suppressed by inhibitory processes.

Endothelium-dependent responses of cerebral blood vessels during chronic hypertension.

Acetylcholine produces less dilatation of pial arterioles in stroke-prone spontaneously hypertensive rats (SHRSP) than in normotensive (WKY) rats. Responses of cerebral vessels to acetylcholine and bradykinin appear to involve different mechanisms. Our first goal was to determine whether responses of pial arterioles to bradykinin are impaired in SHRSP. Diameter of pial arterioles (20-60 microns) was measured using intravital microscopy in WKY rats and SHRSP (9-12 months old). Superfusion of bradykinin (3 x 10(-7) M) dilated pial arterioles by 35 +/- 6% (mean +/- SEM) in WKY rats, but only 21 +/- 3% in SHRSP (p less than 0.05 versus WKY rats). Both nitric oxide (5 x 10(-7) M) and nitroglycerin (10(-5) M) produced similar vasodilatation in WKY rats and SHRSP. Our second goal was to determine whether alteration of postreceptor mechanisms contributes to impairment of endothelium-dependent cerebral vasodilatation in SHRSP. Calcium ionophore A23187 (10(-5) M) produced more vasodilatation in WKY rats than in SHRSP (32 +/- 8% versus 9 +/- 4%, p less than 0.05). Responses to A23187 (10(-5) M) were inhibited by indomethacin (46 +/- 13% versus 15 +/- 5%, p less than 0.05) in WKY rats, whereas responses to A23187 (10(-6) M) were potentiated modestly by indomethacin (-3 +/- 2% versus 4 +/- 2%, p less than 0.05) in SHRSP.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of portal-hepatic osmoreceptors in rats: role of calcium, acetylcholine and cyclic AMP

Osmoreceptors are sensory organs of paramount importance in water and electrolyte balance, yet the mechanisms for their activation are virtually unknown. Peripheral osmoreceptors have been localised in the hepatic portal vein area of rats. We thus superfused the portal adventitia with 0.2 ml of 4% NaCl before and after various pharmacological pretreatments (0.4 ml of 1 mM solutions) of the portal area, while monitoring the neural activity of the hypothalamo-neurohypophysial system. Portal superfusion with verapamil, to reduce Ca-influx, reversibly inhibited the response to osmotic stimuli by up to 50% ( P < 0.0005). Such inhibition (58%; P < 0.0005) was also seen with portal superfusion by atropine. Atropine did not affect hypothalamo-neurohypophysial responses to stimulation of portal bradykinin receptors with 0.2 ml 1 μM bradykinin, and portal superfusion with acetylcholine activated the hypothalamo-neurohypophysial system. The results thus support the hypothesis of a cholinergic neurotransmission linking portal osmoreceptive structures and afferent nerve endings. Diamide, which inhibits water efflux in frog skin, also reversibly inhibited responses to osmotic stimuli by 38% ( P < 0.0005). Pretreatments with trifluoperazine, a calmodulin inhibitor, and cordycepine, an adenylate cyclase inhibitor, diminished responses to osmotic stimuli by 30–45% ( P < 0.005), while cAMP and theophilline potentiated them by 38% ( P < 0.0005). Responses to bradykinin superfusion were reduced 20–30% ( P < 0.05) by both cordycepin and cAMP. The results suggest that portal osmoreceptors release acetylcholine to excite afferent nerves when exposed to an osmotic gradient. The mechanism of this release may be mediated by an efflux of water and an increase of intracellular calcium activity and cAMP.

Spinal substance P transmits bradykinin but not osmotic stimuli from hepatic portal vein to hypothalamus in rat

Peripheral osmo- and bradykinin-sensitive receptors which have been previously localised within the hepatic portal vein area, activate the hypothalamo-neuro-hypophysial system through a neural pathway projecting to the lower thoracic spinal cord. In this paper we attempted to identify the spinal transmitter(s) involved and to answer the question whether osmoreceptors are in fact chemosensitive nociceptors. The portal vein of anesthetised rats was superfused with 0.2ml of 4% NaCl or 1μM bradykinin, and hypothalamo-neurohypophysial responses were measured either electrophysiologically or by radioimmunoassay of arginine vasopressin. Responses to bradykinin, but not to hypertonic saline, were abolished in rats pretreated 2 wks previously with capsaicin s.c., and immunocytochemistry for substance P in these animals showed that substance P was strongly depleted both in the dorsal thoracic spinal cord and in the portal vein. The spinal injection of 8μl 0.1 mM capsaicin at T8–T9 elicited a pronounced hypothalamo-neurohypophysial response, and diminished reversibly the response to bradykinin superfusion of the portal vein. Spinal capsaicin had no effect on responses to hypertonic saline. Similarly, the spinal (T8–T9) injection of 8 μg substance P antagonist, the [ d-Pro 4, d-Trp 7,9,10, Val 8] substance P (4–11), reduced reversibly the responses to bradykinin by about 50% without affecting those to hypertonic saline. The spinal injection of 8 μg substance P, at the same site where substance P antagonist was applied, elicited within 4s a prolonged response (several min). A slightly longer delay between stimulus and neurophysiological response was observed for spinal capsaicin and for bradykinin superfusion. Responses to hypertonic saline superfusion of the portal vein, however, occurred within 1–2 s. The results show that portal vein osmoreceptors are distinct from chemo-sensitive nociceptors, and suggest that substance P may be a spinal mediator for chemo-sensitive portal vein nociceptors. The spinal transmitter for osmosensitive afferents, and the physiological importance of the portal vein area in chemosensation remain to be established.

Morphological and physiological correlation of bradykinin-induced macromolecular efflux

Bradykinin applied topically for 4 min produced marked dose-related increases in the number of fluorescent dextran (mol wt 145,000) vascular leakage sites exclusively from small postcapillary venules--evidence for an increase in macromolecular permeability. The increase in macromolecular permeability was short-lived, making repeated applications possible. The number of bradykinin-induced venular fluorescent dextran leakage sites could be greatly reduced by the simultaneous topical application of isoproterenol, and this antagonism of the increase in macromolecular permeability could be prevented by pretreatment with propranolol. The topical application of papaverine failed to antagonize the increase in the number of venular leakage sites of fluorescent dextran by bradykinin. A continuous 90-min superfusion of bradykinin elicited an initial marked increase in the number of fluorescent dextran venular leakage sites, which then waned after 20-30 min, returning to near control despite the continued superfusion with bradykinin. In canine forelimbs the bradykinin-induced increase in protein efflux, total protein transport, and lymph flow also peaked in approximately 30 min and then waned markedly despite continued local intra-arterial infusions of this agent for prolonged periods. The morphological data from the cheek pouch agrees well with the physiological data from the forelimb, suggesting that an increase in protein efflux in the canine forelimb could be readily explained by an increase in the number of large pores.