Correspondence BRONCHIAL RESPONSIVENESS 10 HISTAMINE IN INFANTS AND OLDER CHILDREN

To the Editor: A recent article by LeSouef and colleagues (1) suggested that nonspecific bronchial reactivity is not increased in younger children and infants, as has been previously reported (2-4), predominantly because of a failure to standardize the dose of inhaled bronchoconstrictor for different-sized children. In presenting their data, the authors state that of the children (n = 30) and infants (n = 45) tested with their methodology, 5 infants and 10 children did not have a provocative histamine concentration that produced a 400'/0 fall (PC40 ) by the end of the challenge, and therefore an extrapolated PC40 value was determined. I would strongly suggest that the use of an extrapolated PC 40 does not have validity. Such a value assumes that the slope of the curve is linear beyond the last tested dose, which is not always true (5). The extrapolated results of these subjects, therefore, cannot be included in the calculation of the mean value for PC40 for the study groups. Certain issues remain unaddressed. For instance, werethe children with extrapolated PC40 values older than children with interpolated PC40 values? The methodology used by these authors does not compare with the methods utilized in our published report on the age effect on bronchial responsiveness (4), making comparisons difficult. We have, however, corrected the bronchial response in our reported subjects (4) using total lung capacity, and have found no influence of differences in total lung capacity on our original observation.

We feel that great caution should be exercised in attempting to make comparisons of levels of responsiveness between children of widely differing age and size. Studies using nebulizers to deliver agonists must make comparisons using inhaled rather than nebulizer solution concentrations. Even then caution is needed as we have little idea of how to control for a multitude of age-related factors that are likely to influence agonist delivery and deposition and an individual's response. These factors include minute ventilation, respiratory rate, inspiratory time, inspiratory flow pattern, airway caliber, and lung volume. Studies such as that of Hopp and coworkers (2), which make comparisons between subjects of differing age on the basis of the dose of agonist needed for a given response, must correct the dose for the size of the subject. In other words, it would be wrong to conclude that if the response for a given dose is greater for a small child than an adult, then the small child has increased airway responsiveness. The problem is in how to size-correct doses; clearly, there is insufficient information available to allow this to be done without unjustifiable assumptions being made. Using total lung capacity to size-correct the dose mayor may not be correct. However, it would seem unreasonable to assume that it is correct, and until the many related methodologic issues are addressed, the question of whether or not there is an age-related change in airway responsiveness in childhood will remain unanswered. . STEPHEN STICK

Research Fellow PETER LESOuEF

Director Department of Respiratory Medicine Princess Margaret Hospital for Children Perth, Western Australia, Australia

J. Hopp, D.O. Associate Professor of Pediatrics Creighton University Omaha, Nebraska RUSSELL

1. Stick SM, Turnbull S, Chua HL, Landau LI, LeSouef PH. Bronchial responsiveness to histamine in infants and older children. Am Rev Respir Dis 1990; 142:1143-6. 2. LeSouef PN, Geelhoed G, Turner OJ, Morgan SEG, Landau LI. Response of normal infants to histamine. Am Rev Respir Dis 1989; 139:62-6. 3. Tepper RS. Airway reactivity in infants: a positive response to methacholine and metaproterenol. J Appl Physiol 1987; 62:1155-9. 4. Hopp RJ, Bewtra AK, Nair NM, Townley RG. The effect of age on methacholine response. J Allergy Clin Immunol 1985; 76:609-13. 5. Neter J, Wasserman W, Kutner MH. Applied linear statistical models. 2nd ed. Homewood, IL: Richard D. Irwin, Inc., 1985;67.

1. Stick SM, Thrnbull S, Chua HL, Landau LI, LeSouef PN. Bronchial responsiveness to histamine in infants and older children. Am Rev Respir Dis 1990; 142:1143-6. 2. Hopp RJ, Bewtra A, Nair NM, Townley RG. The effect of age on methacholine responsiveness. J Allergy Clin Immunol 1985; 76:609-13.

BRADYKININ-INDUCED AIRWAY MICROVASCULAR LEAKAGE AND BRONCHOCONSTRICTION ARE MEDIATED VIA A BRADYKININ B2 RECEP10R

To the Editor: From the Authors: We welcome the opportunity to reply to Dr. Hopp's comments concerning our article (1). We used extrapolation so that we would have a PC40 value for as many subjects as possible. len older children and five infants did not respond to the maximum nebulized concentration of histamine (8 g/L), We therefore calculated PC40 by extrapolation in subjects in whom maximum flow at functional residual capacity fell between 20 and 400'/0 from baseline after receiving 8 g/L, and where necessary corrected for entrainment (PC40[c)). Subjects with extrapolated PC40 values greater than 16 giL (four older children and five infants) were considered nonresponders and assigned a PC40(c) of 8 giL. Six of the older subjects had extrapolated PC40 values between 8 and 16 giL; PC40(c) values calculated from these were all below 8 giL. As explained in our report, allowance for censored data was made by using a Weibull model. The ,exclusionof data obtained using extrapolation does not significantly affect our results; there is still no difference between the level of PC40(c) in infants and older children.

Although Ichinose and Barnes (1) have clearly demonstrated that airway constriction and microvascular leakage, in response to intravenously administered bradykinin (Bk), involve B2 receptors, their interpretation of our work requires clarification. They suggest that the observation (2) that inhaled NPC 349, a Bs-receptor antagonist, had little effect against Bk-induced bronchoconstriction in vivo was due to catabolism of the antagonist by neutral endopeptidase (NEP). This is unlikely because DPhe7-substituted analogs of Bk are resistant to NEP (3) and angiotensin-converting enzyme (ACE) (4). Furthermore, NPC 567, a similar B2 antagonist, caused partial inhibition of Bk-induced bronchoconstriction even when Bk and antagonist were coadministered (inhaled) or administered intravenously 30 s apart (2). It is doubtful that the high doses of antagonists used would be degraded sufficiently to markedly reduce their inhibition of responses to Bk. A limitation of DPhe7 - Bk analogs, including NPC 349 and NPC 567, is their relatively weak affinity for B2 receptors (PKbS ~ 6.00, compared with Bk's pD 2 , ~ 8.(0). Thus, although the molar dose 237

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of NPC 349 used by Ichinose and Barnes was approximately 500~old in excess, constrictions were not abolished. Similarly, we utilized 100 to 300-fold excess doses of B2 antagonists in vivo, but did not abolish responses to Bk (2). Given the low potency of these agents then, it would be difficult to completely inhibit B2 receptormediated responses in vivo. Nevertheless, another possible explanation for the resistance of Bk-induced bronchoconstriction to B2 antagonists exists. Our studies in vitro indicate that pulmonary tissues contain B3 Bk receptors (2). Ichinose and Barnes note that these "... conclusions were based mainly on in vitro studies" (1). Indeed, it would be difficult to classify receptors in any other way. B2 antagonists cause only 600/0 displacement of specific Bk binding in parenchyma and, in tracheal smooth muscle, are inactive. Thus, the B2 receptors in parenchyma, a heterogeneous tissue, may be located on cell types other than smooth muscle. Ichinose and Barnes note that autoradiography suggests there is "... little specific ([3H]-Bk) labeling of airway smooth muscle ... in proximal airways" (1). Undoubtedly, however, contraction of trachealis is caused by Bk acting on receptors on airway smooth muscle. However, B2 antagonists have little effect on these contractions. Furthermore, in tracheal smooth muscle cells, Bk-induced 45Ca2+ efflux is unaffected by B2 antagonists, providing further evidence for B3 receptors (5). It is possible that Bs-receptcr density is too low to visualize autoradiographically as, in binding, there appear to be relatively few B3 receptors (2, 6). Until recently, the proposal for B3 receptors was based on the inability of B1 and B2 antagonists to inhibit Bk's effects. We have found that a novel analog, DArg[Hyp3-Thi 5-OTic 7-Tic8 ] - Bk, is a potent antagonist of ileal B2 receptors and also of Bk-induced contraction and 45Ca2 + efflux in trachea (6). This agent, therefore, is an antagonist of B2 and B3 receptors. Guinea pig pulmonary tissues contain B2 and B3 receptors. B2 receptors may be expressed on sensory nerves, epithelia and vascular endothelial cells (mediating microvascular leakage), whereas B3 receptors are expressed on smooth muscle cells of the conducting airways. We and others observe varying though weak effects of B2 antagonists in guinea pig trachea, and this tissue may indeed contain B1 receptors. However, the inability of well-characterized B2 antagonists, to displace Bk binding (2) or inhibit contraction (2) and 45Ca2+ efflux (5, 6) provided strong evidence for a novel Bk receptor. This is confirmed with DArg[H yp3-Thi5-DTic7-Tic8]-Bk (6). The relevance of B3 receptors is unclear because, in several species including humans, Bk-induced bronchoconstriction has a neuronal reflex origin, with little direct spasmogenic activity on airway smooth muscle. STEPHEN G. FAIlMER, PH.D.

ICI Pharmaceuticals Group Wilmington, Delaware

From the Authors: We classified the Bk receptor involved in airway microvascular leak and bronchoconstriction in guinea pig as a B2 receptor, based on the fact that the B1 receptor antagonist des Argt-Leus-Bk had no antagonistic effect, whereas the B2 antagonist D-Arg-[Hyp3-Thi5. 8D-Phe7]-Bk (NPC 349) caused a similar degree of inhibition in both airway responses. We explained the transient nature of the blockade by enzymatic degradation of this antagonist. Although D-Phe7 Bk analogs such as NPC 349 are resistant to degradation by NEP an~ ACE, they may be broken down by carboxypeptidase N (1), which would be relevant to intravascular administration of this antagonist (2). Perhaps the most likely explanation for the transient inhib~tion is the low affinity of this antagonist for the B2 receptor, as pointed out by Dr. Farmer. Indeed, using such weak antagonists makes it difficult to be confident about the existence of a "B 3receptor" in airway smooth muscle. With the recent development of much more potent B2 antagonists such as HOE 140, which has a pA 2 value of 8 to 9 (3), and the new antagonist mentioned by Farmer, the nature of the Bk-receptor mediating bronchoconstriction will be clarified. As Dr. Farmer points out, the bronchoconstrictor response to Bk in vivo is likely to be mediated indirectly; in guinea pigs the bronchoconstrictor response to intravenous Bk is largely mediated ~ia release of cyclooxygenase products, so that the B2 receptors are likely to be localized to some unidentified cell that releases these products. In contrast, after airway administration of Bk, bronchoconstriction is largely neural in origin, with B2 receptors localized to sensory nerves in the airways (4). In guinea pig airways in vitro, Bk is paradoxically a bronchodilator (because of the release of.pro~taglandins from airway epithelial cells) and is degraded by epithelial NEP (5, 6). After epithelial removal and in the presence of indomethacin, Bk is a weak direct constrictor and this response appears to be mediated by B1 receptors (6). In humans, Bk by inhalation IS a potent bronchoconstrictor in asthmatic patients. Evidence suggests that this is mediated indirectly as cromolyn sodium, nedocromil sodium, and anticholinergic agents all markedly reduce the response (7, 8), whereas Bk is a weak constrictor of airways in vitro. Autoradiographic studies using [3H]Bk confirm that there ~e ~ew Bk receptors on airway smooth muscle of proximal airways In either humans or guinea pigs (9). The classification of Bk receptors that mediate the airway smooth muscle effects of Bk will certainly become easier now that potent antagonists are available, and the cloning of Bk receptors will provide further insights into possible heterogeneity of Bk receptors. Based on studies with weak antagonists such as NPC 349, it would be premature to conclude that Bk receptors in airway smooth muscle differ significantly from other Bk receptors. PETER J. BARNES, D.M., D.Sc., F.R.C.P M. ICHINOSE, M.D.

1. Ichinose M, Barnes PJ. Bradykinin-inducedairwaymicrovascularleakage and bronchoconstriction are mediatedvia a bradykinin B1 receptor. Am

Rev Respir Dis 1990; 142:1104-7. 2. Farmer SO, BurchRM, Meeker SA, WilkinsDE. Evidence for a pulmonary B3 bradykinin receptor. Mol Pharmacol 1989; 36:1-8. 3. WardPEeMetabolismof bradykinin and bradykinin analogs. In: Burch RM, ed. Bradykinin antagonists:basicand clinical research. NewYork: Marcel Dekker Inc, 1990; 147-70. 4. TogoJ, BurchRM, DeHaas CJ, Connor JR, Steranka LR. D-Phe 7-substituted peptide bradykinin antagonists are not substrates for kininase II. Peptides 1989; 10:109-12. S. FarmerSO,Ensor JE, Burch RM. Evidencethat cultured airwaysmooth musclecellscontain bradykinin B1 and B3receptors. Am J Respir Cell Mol BioI 1991; 4:273-7. 6. Farmer SO, Burch RM, Kyle DJ, Martin JA, Meeker SN, Togo J. DArg[Hyp3-Thi5-Dric7-Tiel]-bradykinin,a potent antagonist of smooth muscle BK2 and BK3 receptors. Br J Pharmacol 1991; (In Press).

National Heart & Lung Institute London, United Kingdom 1. Regoli D, Drapeau 0, Rovgro P, et al. Conversionsof kinins and their antagonists with Bi-receptoractivators and blockersin isolated vessels. Eur J Pharmacol 1986; 127:219-24. ~. IchinoseM, BarnesPJ. The effect of peptidaseInhibitorson bradykininInduced bronchoconstriction in guinea-pigs in vivo. Br J Pharmacoll990; 101:77-80. 3. Hock FJ, Wirth K, Albus U, et al. HOE 140 a new potent and long acting bradykinin-antagonist: in vitro studies. Br J Pharmacol 1991· 102:769-73. ' 4. Ichinose M, Belvisi MO, Barnes PJ. Bradykinin-induced bronchoconstriction in guinea-pig in vivo: role of neural mechanisms. J Pharmacol Exp Ther 1990; 253:1207-12. ~. Bramley AM, SamhounMN, Piper PJ. The roleof epithelium in modulatmg the responses of guinea-pig trachea induced by bradykinin in vitro. Br J PharmacoI1990; 99:762-6. 6. FrossardN, StrettonCD,BarnesPJ. Modulation of bradykininresponses in airwaysmooth muscle by epithelialenzymes. AgentsActions 1990; 31:204-9.