Effect of Peptide Histidine Valine on Cardiovascular and Respiratory Function in Normal Subjects

Thorax: first published as 10.1136/thx.43.10.750 on 1 October 1988. Downloaded from

Thorax 1988;43:750-755

Effect of peptide histidine valine on cardiovascular and respiratory function in normal subjects

E R CHILVERS, C M S DIXON, Y YIANGOU, S R BLOOM, P W IND From the Department ofMedicine, Royal Postgraduate Medical School, Hwnmersmith Hospital, London

ABSTRACT Non-adrenergic inhibitory nerves may have an important role in regulating airway calibre. A recently discovered peptide, peptide histidine valine, is a potent relaxer of airway smooth muscle in vitro and has been proposed as a possible neurotransmitter in this tissue. The cardiovascular and respiratory effects of graded infusions of this peptide (2-5-10 pmol kg-' min') have been examined in six normal subjects in a placebo controlled, randomised double blind study. The mean (SEM) peak plasma concentration of peptide histidine valine during the highest infusion rate was 2392 (170) pmol/l, representing a 29 fold increase above the basal concentration. This was accompanied by flushing, a significant increase in heart rate of 28 (3-7) beats/min and skin temperature of 1.80 (0 160) C, but no effect on systolic or diastolic blood pressure. Despite these high plasma concentrations ofthe peptide and the substantial tachycardia and increase in skin blood flow, there was no change in partial expiratory flow at 40% ofvital capacity (Vp4,) or in the airway response to inhaled histamine (geometric PD40 9*37 and 9 73 umol during saline and peptide histidine valinecopyright. infusion respectively). Although these findings provide no support for a physiological role ofpeptide histidine valine in controlling airway function in healthy subjects, important effects oflocally released peptides in the vasoactive intestinal peptide family cannot be excluded. Introdoction precursor peptide (prepro-VIP), with an amino acid sequence corresponding exactly to prepro-VIP-81-122http://thorax.bmj.com/ Non-adrenergic, non-cholinergic nerves have been (fig 1). In vitro peptide histidine valine appears to be recently identified in human airways' and may be more potent than either vasoactive intestinal peptide important in regulating certain aspects of airway or peptide histidine methionine in relaxing guinea pig function, including airway calibre, mucosal gland tracheal smooth muscle'" and is therefore the most secretion, and pulmonary vascular resistance.24 In the active endogenous relaxant of airway smooth muscle absence ofany substantial direct sympathetic innerva- known. tion ofhuman airway smooth muscle' non-adrenergic In normal volunteers vasoactive intestinal peptide, inhibitory nerves form the only neurogenic pathway infused at 6 pmol kg-' mino', has no effect on airway on September 25, 2021 by guest. Protected causing relaxation of airway smooth muscle. The calibre.'2 We have now infused higher doses ofpeptide neurotransmitters of some of these nerves remain histidine valine in normal subjects and investigated the uncertain, although immunocytochemical evidence6 possible physiological role of this peptide in man by and in vitro7 and in vivo studies89 suggest that studying its effects on cardiovascular and respiratory vasoactive intestinal peptide (VIP) and peptide histi- function. dine methionine may play a part. A novel 42 amino acid regulatory peptide, peptide Methods histidine valine, was recently isolated from a human phaeochromocytoma and characterised,'° and sub- We studied six healthy, non-asthmatic subjects (five sequently found in mammalian airways." It is closely male; mean age 30 years) (table). Incremental peptide related to vasoactive intestinal peptide and peptide histidine valine and saline infusions were administered derived from the same on separate days, at the same time ofday, according to histidine methionine, being a randomised double blind design. The two study days were separated by a minimum of one week. Approval Address for reprint requests: Dr E R Chilvers, Department of for this study was obtained from the Hammersmith Thoracic Medicine, Cardiothoracic Institute, London SW3 6LY. Hospital ethical committee and all subjects gave Accepted4 July 1988 written informed consent. 750 Thorax: first published as 10.1136/thx.43.10.750 on 1 October 1988. Downloaded from

Effect ofpeptide histidine valine on cardiovascular and respiratoryfunction in normal subjects 751 PHV five breaths ofeach concentration ofhistamine (doub- ling concentrations from 4 to 64 mg/ml) until a greater than 40% fall in the post-saline Vp, was recorded. From these values the provocative dose of histamine 1 81 122 170 causing a 40% fall in Vp. (PD(,) was interpolated.

4 prepro-VIP P PLASMA PEPTIDE HISTIDINE VALINE CONCENTRATIONS |PHM | |VIP | At each sample time 10 ml ofvenous blood was taken, placed into chilled heparin tubes containing 4000 KIU Fig 1 Peptide histidine valine (PHV), peptide histidine of Trasylol (Bayer UK Ltd, Newbury), centrifuged, methionine (PHM), and vasoactive intestinalpeptide (VIP) and stored at - 20C. Plasma samples were assayed by are all derivedfrom a single 170 amino acidpeptide called means ofa specific radioimmunoassay"' with antibody prepro- VIP. The variable post translationalprocessing required to obtain the individual active peptides is shown with SY1. PHV resultingfrom cleavage at the 88 and 122 amino acid positions as indicated. STUDY PROTOCOL Subjects were studied supine in an air conditioned CARDIOVASCULAR MEASUREMENTS room with a constant temperature of 21°C and had Heart rate recordings were made with continuous refrained from caffeinated drinks on the day of the display electrocardiography (Hewlett Packard, Wal- study. Intravenous cannulas were placed in both tham, USA) and the number of QRS complexes antecubital fossae, and after the subjects had rested for occurring in 60 seconds was counted. Blood pressure 20 minutes baseline cardiovascular and respiratory was measured indirectly with an automatically inflat- measurements were made and blood was taken for ing sphygmomanometer (Dinamap, Critikon, peptide histidine valine assay. Subjects then received a USA). copyright. The mean of two measurements was recorded at each 30 minute saline infusion followed by the active time point. Skin temperature was measured by means peptide or matched saline infusion. The infusions were of a thermistor (Hewlett Packard), with a response prepared in a total volume of50ml containing 10ml of time of seven seconds, placed on the left cheek. the subject's own blood to reduce peptide adherence to the syringe and tubing. The starting concentration of

the infusion was 2 5 pmol kg-' min-' for five minutes; http://thorax.bmj.com/ RESPIRATORY MEASUREMENTS this was followed by 5 0 pmol kg-' min-' for a further Airway calibre was measured by recording airflow at five minutes and a final concentration of 10 pmol/ 60% of vital capacity below total lung capacity, kg-'min' for 30 minutes. Measurements ofheart rate, measured from total lung capacity after a forced blood pressure, skin temperature, and airway partial expiratory flow manoeuvre (Vp,.) with a rolling diameter were made one and five minutes after each seal spirometer (PK Morgan, Chatham) connected to change in infusion rate and at five minute intervals a microcomputer. The mean oftwo measurements was during the highest concentration. Blood samples for recorded. For the histamine challenge test subjects peptide histidine valine assay were taken at 10 minute initially inhaled five breaths of 0 9% normal saline intervals throughout the study period with additional on September 25, 2021 by guest. Protected from a compressed air nebuliser controlled by a breath samples at the end of the 2 5 and 5 pmol kg-' min-' activated dosimeter (Mefar, Brescia, Italy), which was infusion periods. Fifteen minutes from the start ofthe followed after 30 seconds by measurement of Vp4. high dose infusion an inhaled histamine challenge was This procedure was repeated after subjects had inhaled performed. Further cardiovascular and respiratory measurements were made for 15 minutes after the end Characteristics ofthe patients ofthe infusion. VP4 PD40 Two subjects (Nos 3 and 6) were studied on a third Subject Age Weight baseline histamine day in a single blind design with a similar protocol No (y) Sex (kg) Atopy (l/min) (pmol) except that they received a higher dose of peptide 1 29 M 75 - 162 5-1 histidine valine, 20 pmol kg-' min-', which was main- 2 40 M 82 - 122 4-3 tained for 30 minutes. On these two study days the 3 27 M 72 - 172 16-9 4 36 M 76 + 224 9-6 subjects were pretreated with 100 mg atenolol orally 5 22 M 79 + 195 15-4 four hours before the study to limit cardiovascular side 6 24 F 53 + 192 12-4 effects, notably the tachycardia. Limited availability Vp,-flow at 40% vital capacity; PD,0 histamine-airway ofpeptide histidine valine prevented further studies at responsiveness to histamine. this higher dose. Thorax: first published as 10.1136/thx.43.10.750 on 1 October 1988. Downloaded from

752 Chilvers, Dixon, Yiangou, Bloom, Ind 2500 cardiovascular measurements is shown in figure 3. The graded infusion caused a progressive increase in heart 0 rate, which reached a maximum of 28 (3-7) beats/min E (p < 005) immediately before the histamine CC 2000- challenge. The tachycardia first achieved statistical

- significance five minutes after the start of the 5 pmol I kg' min-' infusion, when the mean plasma peptide histidine valine concentration was 438 pmol/l. The 0~ E1500 - increase in heart rate appeared to plateau with increas- (Uco ing plasma concentrations ofthe peptide, little further increase occurring with plasma concentrations over 1500 pmol/l. Peptide histidine valine caused a progres- 1000 - sive increase in skin temperature, with a maximum rise of 1-80 (0-160) C (p < 0-05) 15 minutes after the startof the 10 pmol/l infusion, before the administration of 500 - 2.5 - .1 -5 cI 0 J EC- 1.5- 10 - (U)

5.] 0 .V 2.5 - E 0 ~~~~~0.5- -aE 0 0- -10 0 10 20 30 40 time (min) -0.5. copyright. Fig 2 Plasma peptide histidine valine (PHV) 120 concentrations (means with standard errors) achieved during I ;I I_; I PHV infusion. The shaded area indicates the infusion protocol. PHV was measured as PHV immunoreactivity as = 100 - outlined in the text. 0) http://thorax.bmj.com/ QL1D1E

O , 80 - DATA ANALYSIS Results in the text are expressed as means with 60 standard errors in parentheses. For statistical analysis we used analysis ofvariance ofrepeated measurements 100 - and Student's t test. Vp4o data are expressed as _ 90 - A a) ______1~~~~~~~--

geometric mean values with 95% confidence intervals on September 25, 2021 by guest. Protected 1- .: and compared by means of the Mann-Whitney U test tE 80 - after logarithmic transformation. co D. I- 70 I ___-d Results -

E Subjects tolerated the infusions well and all received the highest concentration of histidine valine of 10 pmol kg- min-'. Mild flushing was the only reported E mean a. side effect. The (SEM) plasma peptide histidine -10 0 5 10 20 30 40 valine concentration increased from a basal value of 81-5 to 2392 pmol/l 30 minutes after the start time (min) (8) (170) Fig 3 Cardiovascular effects ofinfusedpeptide histidine ofthe 10 pmol kg-' min-' infusion (fig 2). A significant valine (PHV) (-)andsaline control (A) (means with increase in plasma peptide histidine valine concentra- standard errors). Bloodpressure values during the saline tion was seen five minutes after the start ofthe 2-5 and infusion are not included as they are virtually identical to 5*0 pmol kg-' min7' infusion rates (p < 0-05). those shownfor the PHV infusion day. The arrow indicates The effect ofinfused peptide histidine valine on the the start ofthe histamine challenge. Thorax: first published as 10.1136/thx.43.10.750 on 1 October 1988. Downloaded from

Effect ofpeptide histidine valine on cardiovascular and respiratoryfunction in normal subjects 753 heart rate and skin temperature but had no effect on 200 4 systolic or diastolic blood pressure or on airway calibre in these normal subjects. The plasma levels of .-- 0. the peptide achieved in this study are some 20 fold oE 180 - higher than the plasma concentrations of vasoactive Q C ,, *- intestinal peptide produced by infusion in a similar *> study and represent a 29 fold increase over basal oE 160 - peptide histidine valine concentrations."2 This reflects Q._ the relatively long half life of the peptide when 140 - infused.'3 Despite these high plasma concentrations and the potency ofpeptide histidine valine in relaxing airway smooth muscle in vitro (PHV > VIP > adren-

- 120J aline),10 we have been unable to find any effect on YHISTAMINE resting airway calibre or histamine responsiveness in E 10 - normal subjects at this dose. The large changes in Vp4 >. 5- values seen after the administration of a beta agonist 2.5- and the small intrasubject variability of the a--s - measurement indicate that this measurement is a -10 0 5 10 20 30 40 sensitive index of airway calibre and that small time (nnin) changes should have been detected. These findings are in keeping with a similar lack of Fig 4 Effect ofpeptide histidine valine (PHV)*(O) and effect of infused vasoactive intestinal peptide on saline control (A) infusion on airway calibre (Vp., means airway function in normal subjects,'2 although a small with standard errors) before histamine c)hallenge (indicated by the arrow). bronchodilator response has been reported in asthmatic subjects.8 The dose of vasoactive intestinal copyright. histamine. Despite these substantiEal effects on heart peptide given by infusion is limited by the reduction in rate and skin temperature no changge was observed in systolic blood pressure, and in the above study'2 either systolic or diastolic blood pressure during the infusion of the peptide (fig 3). Peptide histidine valine caused no change in Vp4o at any infusion rate in any subject (fig 4). Inhalation of - http://thorax.bmj.com/ 800 jig isoprenaline from a metered dose inhaler by the 20 - same subjects caused an increase in mean Vp(, of 57% E (8%). The mean coefficient of variation of the Vp, 0) values obtained during placebo infusion was 11% C within subjects. All subjects had a greater than 40% E 10- fall in 'Vp, during the histamine challenge. The geometric mean PD40 histamine was 9-37 .mol (95% +._

confidence interval 5-85-15-01) during the saline on September 25, 2021 by guest. Protected infusion and 9 73 umol (6-2-15-26) during the peptide 0 infusion (fig 5). This difference was not significant. o 5 - The two subjects who received peptide histidine 0I valine 20 pmol kg-' min7' had increases of 27% and 24% in Vp4, over baseline values after 15 minutes of this infusion. This compared with no increase and a 13% increase 15 minutes from the start ofthe 10 pmol kg' min-' infusion on the previous study day. PD,, 2] histamine values were increased from 12-3 and 16-9 I I umol during the 10 pmol kg-' min-' infusion to 22-5 Saline PHV and 24-3 umol during the 20pmol kg-' min-' infusion in the two subjects. (1 0 pmol kg-' min-') Figs Histamine responsiveness (PD,o) during saline Discussion infusion and 15 minutesfrom the start ofthepeptide histidine valine (PHV) infusion (JO pmol kg-' min-'). Each point Infusion ofpeptide histidine valine in doses of up to 10 represents the valuefor one subject and the horizontal lines pmol kg-' min-' caused a dose dependent increase in indicate geometric mean values. Thorax: first published as 10.1136/thx.43.10.750 on 1 October 1988. Downloaded from

754 Chilvers, Dixon, Yiangou, Bloom, Ind plasma concentrations of only 34-6 pmol/l were major circulating peptide in patients with VIPomas, achieved with an infusion rate of 6 pmol kg' min-'. with concentrations 10 times higher than those of Nevertheless, there is now considerable evidence from vasoactive intestinal peptide orpeptide histidine meth- in vitro studies suggesting that this peptide may ionine, and it may be responsible for the profuse function as a neurotransmitter of non-adrenergic diarrhoea and other clinical features seen in patients inhibitory nerves in man.'4 The failure to detect an with these tumours.'6 effect on airway tone when peptide histidine valine or The possible role of peptide histidine valine as a vasoactive intestinal peptide is infused in man may be neurotransmitter of non-adrenergic nerves in airway due to the problem of access of the peptide to its smooth muscle needs further investigation. Although airway receptor. This may be due in part to rapid no significant effect on airway calibre or histamine enzymatic breakdown in the tissues. This should be responsiveness was seen at infusion rates up to 10 pmol less with peptide histidine valine as its plasma halflife kg-' min-', higher doses may result in a small increase is about 50 times greater than vasoactive intestinal in both these measurements. Our study does not rule peptide in man.'3 In addition, extremely high circulat- out an effect of locally released peptide histidine ing concentrations may be necessary to achieve con- valine. The close relationship of this peptide to centrations of the peptide at airway receptors com- vasoactive intestinal peptide and peptide histidine parable to those that are possible after local peptide methionine, its powerful pharmacological actions in release from nerve terminals. Inhaled vasoactive intes- vitro, and its localisation in airway tissue indicate that tinal peptide has been shown to afford protection it may have a role in regulating airway function. against histamine induced bronchoconstriction only in those with atopic asthma9 and thus the possibility of an effect of peptide histidine valine administered by this route, particularly in individuals with asthma, cannot be discounted. References To examine the possibility that an airway effect 1 Richardson JB, Beland J. Nonadrenergic inhibitorycopyright. might occur with a higher dose of peptide histidine nerves in human airways. J Appl Physiol 1976;41: valine, two subjects were studied on a third day with a 764-71. higher dose, 20 pmol kg-' min-', of infused peptide 2 Davis C, Kannan MS, Jones TR, Daniel EE. Control of histidine valine and a similar protocol. Although this human airway smooth muscle: in vitro studies. J Appl study was uncontrolled, the increase in Vp0 and PD., Physiol 1982;53:1080-7. histamine in both subjects suggests that infused pep- 3 Peatfield AC, Richardson PS. Evidence for non-choliner- http://thorax.bmj.com/ tide histidine valine has the potential, at higher doses, gic, non-adrenergic nervous control ofmucus secretion to cause bronchodilatation. into the cat trachea. J Physiol 1983;342:335-45. of infused peptide his- 4 Hamasaki Y, Said SI. Evidence for a non-adrenergic, The cardiovascular effects non-cholinergic inhibitory nervous system in cat pul- tidine valine in producing a considerable tachycardia monary artery [abstract]. Clin Res 1981;29:550A. and increase in skin temperature, but no change in 5 Dodge JM, Satchell DG. Adrenergic and non-adrenergic blood pressure, are intriguing. In this respect the effect inhibitory nerves in mammalian airways. J Auton Nerv of peptide histidine valine is quite distinct from Syst 1982;5:83-99. histidine 6 Dey RD, Shannon WA, Said SI. Localization of VIP- vasoactive intestinal peptide and peptide on September 25, 2021 by guest. Protected methionine, which at much lower plasma concentra- immunoreactive nerves in airways and pulmonary tions, and for the same degrees ofchange in heart rate vessels of dogs, cats and human subjects. Cell Tissue substantial falls in Res 1981;220:231-8. and skin temperature, produce 7 Said SI, Kitamura S, Yoshida T, Preskitt J, Holden LD. diastolic and to a lesser extent in systolic blood Humoral control of airways. Ann NY Acad Sci pressure.'3 A direct chronotropic effect of peptide 1974;221:102-14. histidine valine on the heart has been proposed,'3 but 8 Barnes PJ, Dixon CMS. The effect of inhaled vasoactive its apparent lack ofeffect on arteriolar resistance while intestinal peptide on bronchial reactivity to histamine it is dilating skin vessels is unexplained. in humans. Am Rev Respir Dis 1984;130:162-6. The physiological role of peptide histidine valine is 9 Morice A, Unwin RJ, Sever PS. Vasoactive intestinal uncertain. In addition to its effects on airway smooth peptide causes bronchodilatation and protects against muscle in vitro it is a potent relaxer of guinea pig histamine-induced bronchoconstriction in asthmatic muscle and reduces the force and subjects. Lancet 1983;ii: 1225-6. gastric smooth 10 Yiangou Y, Di Marzo V, Spokes RA, Panico M, frequency of spontaneous uterine contractions in the Morris HR, Bloom SR. Isolation, characterization and rat.'0 It has been identified in mammalian nasal pharmacological actions ofpeptide histidine valine 42, mucosa, stomach, genitalia, and airways." '5 In man, a novel prepro-vasoactive intestinal peptide-derived normal circulating concentrations of the peptide in peptide. JBiol Chem 1987;262:14010-3. the resting state are around 80 pmol/l. It is the 11 Ghatei MA, Springall DR, Richards IM, et al. 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Effect ofpeptide histidine valine on cardiovascular and respiratoryfunction in normal subjects 755 Regulatory peptides in the respiratory tract of Macaca 14 Anonymous. NANC nerves in airways [editorial]. Lancet fascicularis. Thorax 1987;42:431-9. 1986;ii: 1253-4. 12 Palmer JBD, Cuss FMC, Warren JB, Blank M, 15 Yiangou Y, Requejo F, Polak JM, Bloom SR. Charac- Bloom SR, Barnes PJ. Effect of infused vasoactive terization of a novel prepro VIP derived peptide. intestinal peptide on airway function in normal Biochem Biophys Res Commun 1986;139:1142-9. subjects. Thorax 1986;41:663-6. 16 Yiangou Y, Williams SJ, Bishop AE, Polak JM, 13 Gill JS, Yiangou Y, Chrysanthou BJ, et al. Peptide Bloom SR. Peptide histidine-methionine immuno- histidine valine: cardiovascular actions and phar- reactivity in plasma and tissue in patients with vaso- macokinetics in man differ from vasoactive intestinal active intestinal peptide-secreting tumours and watery peptide and peptide histidine methionine. Clin Sci (in diarrhoea syndrome. J Clin Endocrinol Metab press). 1987;64: 131-9. copyright. http://thorax.bmj.com/ on September 25, 2021 by guest. Protected