The effect of a selective ar,-adrenergic

receptor antagonist (midaglizole) on the cyclic

3’,5’-adenosine monophosphate production in
human mononuclear cells

Haruhito Sugiyama, MD, Hidefumi Miyagawa, MD, Chiharu Okada, MD,

Russell J. Hopp, DO, Againdra K. Bewtra, MD, and Robert G. Townley, MD
Omaha, Neb.

A selective cu,-adrenergic antagonist, midaglizole, has been recently reported to have efJicacy in
patients with asthma. To understand the mechanisms, we investigated the effect of midaglizole
on the cyclic 3’,5’-adenosine monophosphate (CAMP) production in human mononuclear cells
(MNCs). MNCs were separated by a histopaque gradient from 10 normal subjects and IO
subjects with asthma. CAMP was measured by RIA kits. Mia’aglizole (10 pm01 IL) significantly
enhanced isoproterenol-induced CAMP production in both groups, although midaglizole (from 1
to 100 pmollL) did not increase the CAMP production by itself. The percent increase in CAMP
was more in subjects with asthma (183.8%) than in normal subjects (140.4%); however, the
absolute increase was not different. Platelet-activating factor has been demonstrated to inhibit
pagonist-induced CAMP production in several mammalian tissues, including human MNCs.
Midaglizole also prevented platelet-activating factor-induced desensitization of the CAMP
response to isoproterenol in h4NCs. These findings suggest that midaglizole may be a useful
additional agent for the therapy of bronchial asthma through an enhancement of the CAMP
production. (J ALLERGY CLINIMMUNOL1992;89:722-9.)
Key words: Bronchial asthma, midaglizole, &adrenergic antagonist, cyclic 3’,5’-adenosine
monophosphate, mononuclear cells, platelet-activating factor

Although the precisepathophysiologicmechanisms

of BA remain unknown, a functional imbalanceof CL- Abbreviations used
and P-adrenergic-receptorresponseshas been con- CAMP: Cyclic 3’,5’-adenosine
monophosphate
sidered as one of the fundamental abnormalities in MNC: Mononuclear cell
asthma.‘3’ The P-adrenergicresponsivenessof leu- PAF: Platelet-activating factor
kocytes from subjectswith asthmais significantly de- BA: Bronchial asthma
Gs protein: Stimulatory subunit of the guanine nu-
pressedcomparedwith that of normal subjects.3,4 The
cleotide regulatory protein
function of P-adrenergicreceptorson the cell surface Gi protein: Inhibitory subunit of the guanine nu-
is likely to be modulated by a number of factors5 cleotide regulatory protein
Recently, we demonstratedthat PAF decreasedthe HBSS: Hanks’ balanced salt solution
density of p-adrenoceptorsin the human lung and
reducedthe responsivenessof P-adrenoceptorsto p-
agonistsin human tracheaand MNCS.~*’
Szentivanyi et al.* also consideredthe possibility
From Cmighton University, Allergic Disease.Center, Omaha, Neb. of hypersensitivear-adrenergicreceptors in asthma.
Supported in part by a grant horn Daiichi PharmaceuticalCo., There is evidencein some subjectswith asthmathat
Ltd., Tokyo, Japan. the administration of an a-agonist, such as methox-
Received for publication July 8, 1991. amine, provokes airway narrowing99I0 and that sub-
Revised Nov. 18, 1991. jects with asthma may respond to treatment with o-
Accepted for publication Nov. 19, 1991.
Reprint requests:Hamhito Sugiyama, MD, Cmighton University, adrenergicantagonists,such as phentolamineand thy-
Allergic DiseaseCenter, 2500 California St., Omaha, NE 68178. moxamine.“’I2In contrast,thereare somestudiesthat
l/1/35070 reveal o-antagonists have no effect on pulmonary
722
VOLUME 89
NUMBER 3
function in subjectswith asthma.‘3“sIt still remains
controversialwhethera-adrenergic(especiallyol,-ad-
renergic)hypersensitivitymay play an importantrole
in the pathogenesis of BA. Studieswith moreselective
ol,-adrenergic-receptor antagonistsare necessaryto
elucidatethe exact role of a,-adrenergicreceptorsin
asthma.
M idaglizole (2-[2-(4,5-dihydro-lH-imidazol-Zyl)-
I -phenylethyl] pyridine dihydrochloride sesquihy-
drate), a new selectivea,-adrenergic-receptor antag-
onist, hasbeenrecentlydevelopedin Japan.I6M idag-
lizole hasbeenintroducedas a new oral hypoglycemic
drugthroughits antagonismof a,-adrenergicreceptors
on pancreaticbeta cells.” M idaglizole has beenre-
cently demonstrated to have a bronchodilatingeffect
in moderateto severeBA. I7To understandthe mech-
anism of the effect of m idaglizolein asthma,we in-
vestigatedthe effect of m idaglizoleon the CAMP pro-
duction in human MNCs. W e also investigated
whether m idaglizole preventsPAF-induceddesensi-
tization of the CAMP responseto a P-agonist in
MNCs.
MATERIAL AND METHODS
Chemicals
Chemicals were obtained from the following sources:
ethylenediaminetetraacetic acid, trichloroacetic acid, ethyl
ether,andWright’s stain solutionfrom FisherScientific Co.,
Orangeburg,N.Y.; dextran, Histopaque,Giemsa-stainso-
lution, trypanblue solution, humanalbumin, 1-epinephrine,
and l-isoproterenol from Sigma Chemical Co., St. Louis,
MO.; HBSS from Gibco Laboratories,GrandIsland, N.Y.;
Turk blood-diluting fluid from Ricca Chemical Co., Ar-
lington, Texas; Cl6 PAF from Bachem Laboratory, Inc.,
Torrance,Calif. ; midaglizole from Daiichi Pharmaceutical
Co. Ltd., Tokyo, Japan;and ‘251-labeled CAMP RIA kit from
New EnglandNuclear-DuPont,Boston, Mass.
Subjects
Ten normal healthy subjects(all were male) and 20 sub-
jects with mild asthma(all were male) participatedin this
study. The meanageof the normalsubjectswas 27.4 + 1.2
years (mean + SEM), and of the patients with asthma,
30.2 f 1.4 years. Normal subjectshad no history of atopy
and took no medicationsfor at least 1 month. All subjects
with asthmahad a history of recurrentepisodic dyspnea,
wheezing, and chest tightnessthat improved with a bron-
chodilator.Subjectswith asthmawere all asymptomaticand
had not required maintenanceP-agonists,corticosteroids,
or any antiallergic drugs for at least 2 weeks before their
participation in this study. All subjectswere selectedto
excludehypertension,endocrinediseases,smoking, or psy-
chiatric disorders. All participantsrefrained from coffee,
tea, cola beverages,alcohol, and strenousexercisefor 24
hours before the study. Venousblood was drawn in the
morning to avoid circadian changesin CAMP. Informed
Effect of midaglizole on CAMP productinv 723
consentwas obtainedfrom all participants,and this stud)
was approvedby the CreightonUniversity Institutional Re-
view Board.
Cell preparation
Forty milliliters of venous blood was drawn into tube\
with 1.S% ethylenediaminetetraacetic acid. Blood was in-
cubated with 6 % dextran in normal saline for 30 to 60
minutesat room temperature.The plasma-leukocyte fraction
was decantedand diluted with HBSS without Ca’ +!Mg~
and washedtwice by low-speedcentrifugation (130 R for
10 minutesat 4”C) to removeplatelets.MNCs wereisolated
by density-gradientcentrifugationon Histopaque(density,
1.077) (700 8 for 35 minutesat room temperature).MNCs
were washedtwice in cold HBSS with low-speedcentrif-
ugation. The cell count was performed with a hemocy-
tometer with Turk stain, and the cell population was cval-
uatedby cytospinslidesstainedwith Wright’s Giemsa stain.
The MNC fraction usually containedabout 86% lympho-
cytes and 12% monocytes,and the polymorphonuclearcell
contaminationwas <2%. The ratio of platelets to MNCs
was <O. 1. There was no difference in the cell differential
and platelet contaminationbetweenboth groups. The via-
bility of MNCs was >97% with trypan blue dye exclusion
method.Finally, MNCs (1 x 10’ml) were resuspended in
HBSS with Ca* ’iMg _ _ and0.03% humanserumalhumin.
CAMP assay
Incubations(in duplicate) were performedin 0.5 ml of
final volumesin glasstubesat 37”C. The cells were prein-
cubated,with or without, midaglizole ( 1 to l(K) pmol/L)
for 10 minutes, and then appropriateconcentrationsof iso-
proterenol, epinephrine,and/or PAF were added.The in-
cubationwas allowed to continuefor an additional 10 min-
utes. The reaction was terminatedby addition of 0.5 ml of
12%trichloroaceticacid, followed by centrifugationat 1500
g for 10 minutes at 4” C. The supematants were purified
by ether extraction and kept at - 70”C until cAMP assay
was performed. CAMP was determinedwith ‘“K-labeled
CAMP RIA kits.
Statistical analysis
All the values are representedas mean 2 SEM. Statis-
tical analysiswas performedby an analysisof variancetest
for unpairedobservationsor W ilcoxon’s test for pairedob-
servations;p valuesco.05 were consideredstatistically sig-
nificant.
RESULTS
Although m idaglizole(from 1 to 100 p m 0 1/ L) did
not increasethe CAMP productionby itself in both
normal subjectsand groups with asthma(Fig. I),
m idagiizoleat 10 p,mol/L significantlyenhancediso-
proterenol-induced CAMP productionin both groups
(Fig. 2). W ith 1 p,mol/L of isoproterenol,the CAMP
content increasedfrom 11.7 t 1.3 to 15.2 2 1.9
p M in normal subjects and from S.9 x 1.O to
724 Sugiyama et al. J. ALLERGY CLIN. IMMUNOL.
MARCH 1992

10 r nephrine-inducedCAMP production synergistically

0 Normals (Table II). Although midaglizole had a tendency to
gQ l Asthmatics
1
enhanceCAMP accumulationinducedby epinephrine
more than by isoproterenol,it did not reachstatistical
r 6-
significance.
5 7- We have recently reportedthat PAF preventedthe

b_V’
CAMP responseto isoproterenolin human MNCs.’
a 6- As illustrated in Fig. 5, PAF at 0.1 and 1 kmol/L
significantly decreasedisoproterenol-inducedCAMP
- 5-
E
production in MNCs from normal subjects and sub-
s 4-
jects with asthma.Therewas no significant difference
in the responseto PAF betweennormal subjectsand

!r.;
5
0 3-
!$ *-
0 l-
I icantly (p < 0.05) increased isoproterenol-induced
oki7i ; llo 100
Concentration of midaglizole (uM)
FIG. 1. Effect of midaglizole on CAMP production in human
MNCs. No significant increase in CAMP in normal subjects
and in subjects with asthma. No statistical difference be-
tween the two groups.
9.1 t 1.7 pM/ lo6 MNCs in subjects with asthma
(TableI). In subjectswith asthmathe CAMP response
to isoproterenol,with or without midaglizole, is lower
than in norrnd subjects, as presentedin Table I. This
finding is consistent with previous studies.3.4, ’ An
enhancementis illustrated in Fig. 3 of the CAMP pro-
duction by midaglizole in the presenceof isoproter-
enol, which was obtainedby subtractionof the CAMP
productioninducedby midaglizole alone. In the pres-
ence of 1 pmol/L of isoproterenol,midaglizole sig-
nificantly enhancedthe CAMP production in a dose-
dependentmannerin both groups.
The CAMP production induced by midaglizole in
the absenceor presenceof isoproterenolwas obtained
by subtractionof the basal CAMP level (Fig. 4). Mi-
daglizole alone (10 Fmol/L) increased CAMP of
0.8 +- 0.5 pM in normal subjects,and0.5 + 0.3 pM
in subjectswith asthma.When midaglizole was com-
bined with isoproterenol,however,a synergisticeffect
on the CAMP productionwas observedin both groups.
In subjects with asthma the CAMP production in-
creasedfrom 4.0 + 0.6 to 7.2 4 1.1 pM, and in
normal subjects, from 8.1 f: 1.0 to 11.5 -t 1.3 pM.
The value of percentincreaseon the CAMP production
in subjects with asthma (183.8% ? 18.9%) was
significantly higher than in normal subjects
(140.4% 2 13.8%). Midaglizole also enhancedepi-
subjects with asthma. Midaglizole, at 10 and 100
pmol / L, preventedPAF ( 1 p,mol/ L)-induced desen-
sitization of the CAMP responseto isoproterenolin
MNCs from both groups (Table III). In subjectswith
asthma, midaglizole, at 10 and 100 pmol/L, signif-
CAMP production, even in the presenceof PAF. In
contrast, normal subjectshad a significant increasein
CAMP at 100 p,mol/ L of midaglizole in the presence
of PAF. This finding suggestedthat subjects with
asthmarespondto midaglizole at lower concentrations
than normal subjects.
DISCUSSION
Several investigators have demonstratedthe effi-
cacy of cu-adrenergic-receptorantagonists,such as
phentolamineand thymoxamine, in patientswith ex-
ercise-inducedor allergen-inducedBA.“* I2 The com-
bination of isoproterenoland either phentolamineor
thymoxamineproducedmarked improvementin pul-
monary function, significantly more improvement
than isoproterenolalone.‘*,I9 In supportof theseclin-
ical findings are the observationsthat phentolamine
or thymoxamineenhancedthe CAMP responseto iso-
proterenol in leukocytes from normal subjects and
subjectswith asthma.*‘.*’In contrast, there are some
studiesthat reveal a-antagonistswere not effective in
subjects with asthma.‘3-‘5According to the observa-
tion by Spector,13when phentolaminewas adminis-
tered by inhalation to 68 subjects with asthma, only
16% had bronchodilatationand 12% had broncho-
constriction. Theseclinical and experimentalfindings
suggest the existence of cw-adrenergic hyperrespon-
sivenessin a subsetof the populationwith asthmaand
suggesta-receptorblockademay improve the clinical
condition of subjectswith asthmathroughan elevation
of the CAMP production. When the efficacy of OL-
adrenergicantagonistsare considered, as described
above,thesea-antagonistsmay have otherproperties,
such as cr,-antagonisticand antihistamineactivity, in
addition to a,-antagonisticactivity. To clarify the sig-
nificance of ol,-adrenoceptormechanismsin asthma,
dOLUME 89 Effect of midaglizole on CAMP uroducfior; 725
YUMBER 3

A Normals B Asthmatics

“?ik?W “5izY t Obl ; 100

Concentration of isoproterenol (uM) Commtratian of isopfoteremt (LM)
FIG. 2. A, Effect of combination of midaglizole and isoproterenol on CAMP production in MNCs
from normal subjects. 6, Effect on CAMP production in MNCs from subjects with asthma. Con-
centration of midaglizole used was 10 PmollL; *p < 0.05, compared with value in control group
(isoproterenol alone) by Wilcoxon’s test.

TABLE 1. Summary of CAMP production in MNCs induced by isoproterenol and midaglizole

CAMP content #k+lIloL MNCt)

Subjects

With
Normal asthma
Conditions (N = IO) (N = IO)

Basal value 3.7 + 1.2 1.8 + 0.9

Is0
I ymol/L 11.7 t 1.3 5.9 I I.0
100 ~mol/L 11.8 I 1.3 6.5 -+- 1.2
Mid 10 p,mol/L 4.5 i 1.2t 2.3 II- 0.81
Mid plus Iso 1 bmol/L 15.2 r 1.91: 9.1 ” 1.73
Mid plus Iso 100 u m o l/L 14.6 2 1.3$ 8.1 1_ l.ZEi

Iso. Isoproterenol;Mid, midaglizole; NS, not significant.

Comparedwith the value inducedby isoproterenolalone in the samegroup by W ilcoxon’s test:
*p values were obtainedby analysisof variancetest.
?p, not significant, comparedwith a basal value by W ilcoxon’s test.
tp c 0.05
Ejp d 0.02.

more preferential and specific a,-antagonistshave antagonistthanyohimbineandlacks antihistamineac-

beendeveloped.
M idaglizole is demonstratedto be a specific 02-
adrenergic-receptor antagonistthroughstudieson in-
sulin releasefrom pancreaticislets and radioligand
binding assayin rat cerebralcortex membranes.16. ”
M idaglizole is a more preferentialand specific 02-
tivity.22,23W h e n m idaglizolewas combinedwith iso-
proterenol, m idaglizole significantly enhancedthe
bronchodilatingeffect of isoproterenol .23~id&gli~ole
was also effective in patientswith moderatcto severe
BA when it was addedto conventionaltreatment,*7
In this study, we demonstrated that m idaglizoleen-
726 Sugiyama et al. J. ALLERGY CLIN. IMMUNOL.
MARCH 1992

* production is mediated via cl,-adrenergicreceptors.

The synergistic effect on CAMP production is also
demonstratedwith another a-antagonists, such as
phentolamineand thymoxamine.*‘,2’The effect of
phentolaminemight be due to /3-agonisticactivity as
,-i;
well as an a-blockade.20However, we consideredo-
blockadeas the main reasonfor the synergistic effect
of midaglizole becausemidaglizole has been dem-
onstratednot to have P-agonistic activity.25
In considering the effect of a-antagonistson the
;---i-; CAMP production in MNCs, the problem of platelet
contaminationin the MNC fraction is significant. Lad
et a1.26reportedthat there was no a,-receptorbinding
0 Normals in MNCs from normal subjects, althoughlow levels
l Asthmatics of a,-adrenoceptorbinding werefound in MNCs from
subjectswith mild asthmawith highly purified MNCs
1 1 (platelet contamination, <I%). It has been reported
I ’
OB85sI 1 10 100 that there was no difference in density and affinity of
platelet a,-receptorsor in platelet CAMP responsesto
Concentration of midaglide Mt) stimulation by a-agonists between normal subjects
FIG. 3. Enhancement of CAMP production by midaglizole
and subjects with asthma.‘,27Although the platelet
in the presence of isoproterenol in normal subjects and contaminationin the MNC fraction was <9% in this
subjects with asthma. Concentration of isoproterenol study, platelets may play a role in the effect of mi-
used was 1 PmollL. CAMP increase by midaglitole was daglizole on the CAMP production in MNCs from
obtained by subtraction of CAMP production induced by normal subjects. The finding that a synergistic effect
midaglizole alone; *p < 0.05, compared with basal value
without midaglizole by Wilcoxon’s test. Significant differ-
of midaglizole on isoproterenol-inducedCAMP pro-
ences (p < 0.05) between two groups at all concentrations duction resulted in a higher percent increasein sub-
of midaglizole by analysis of variance test. jects with asthmathan in normal subjectsmay, how-
ever, be accountedby the presenceof increasedor
hypersensitivecr,-adrenoceptors in MNCs from sub-
hancedisoproterenol-induced CAMP productionin hu- jects with asthma,comparedwith that of normal sub-
man MNCs from normal subjects and subjects with jects.
asthma, although it did not increasethe CAMP pro- The adenylatecyclase system that producesCAMP
duction by itself. Moreover, this synergistic effect of in cell surfaceis regulatedby both a stimulating factor
midaglizole on the CAMP production resulted in a (Gs protein) and an inhibitory factor (Gi protein).28
higher percent increasein subjects with asthmathan Activation of o,-adrenergicreceptorsinhibits the cat-
in normal subjects. This synergistic effect of midag- alytic unit of adenylatecyclase via activation of Gi
lizole was also found on epinephrine-inducedCAMP protein (releaseof Gi o-subunits). Therefore,a,-ad-
production. We expectedthat midaglizole would be renergic-receptorantagonistsare suggestedto act on
more potent on epinephrine-inducedCAMP produc- adenylatecyclase througha,-adrenergicreceptorsand
tion than on isoproterenol-inducedCAMP production Gi protein. The exact mechanism by which an CX-
becauseepinephrineis known to have more a,-ad- blockaderesults in the enhancementof isoproterenol-
renergic activity. Although midaglizole had a ten- induced CAMP production in MNCs remains un-
dency to enhanceCAMP accumulation induced by known. It has been postulated, however, that there
epinephrinemore than that inducedby isoproterenol, may be a direct competition betweenGs and Gi (Y-
we could not find any significant differencesbetween subunits on the adenylatecyclase and that a,-antag-
thesetwo adrenergicagents. However, Sakai et al.% onists might result in the enhancementof the CAMP
recently reportedthat BHT-933 (specific cr,-adrener- synthesisvia inhibition of cY2-receptors and Gi o-sub-
gic agonist) suppressedisoproterenol-inducedCAMP units release.’Additional studiesare necessaryto in-
production from human leukocytes and that midag- vestigatethe exact mechanismof the synergisticeffect
lizole preventedthe effect of BHT-933 on isoproter- of cw,-antagonists.
enol-inducedCAMP production. This study suggests We also demonstratedin this study that midaglizole
that the synergistic effect of midaglizole on CAMP preventedPAF-induceddesensitizationof the CAMP
VOLUME 89 Effect of midaglizole on CAMP produc!!:>n 727
NUMBER 3

Normals Asthmatics

m MiU(lOuM) Iso(luM) 63 Mrd+lso

FlG. 4. Effect of midaglizole on isoproterenol-induced CAMP production in MNCs. Concentrations

of midaglizole and isoproterenol used were 10 and 1 FmoliL, respectively. Each value was
subtracted by a basal CAMP level; **p < 0.01, compared with value induced by isoproterenol
alone in same group by Wilcoxon’s test. Values of percent increase on CAMP production in
subjects with asthma and in normal subjects were 183.8% t 18.9% and 140.4% 1 13.8%, re-
spectively (p < 0.02, by analysis of variance test).

responseto isoproterenolin MNCs from normal sub- 100%

T
jects and subjectswith asthma.PAF has beendem-
onstratedto inhibit the prostaglandinE,-inducedrise
in CAMP in rabbit platelets,” prostaglandinE,- and
salbutamol-induced CAMP productionin guineapigs
i! i:-;-a:-:I,
alveolar macrophages,iO and isoproterenol-induced
CAMP production in human MNCs.’ cl-Adrenergic
I
antagonists,phentolamineand phenoxybenzamine, V * I
arereported to specificallyinhibit PAF-inducedplate- 3
50% -
let and neutrophil aggregationand degranulation.3’ E
B
Recently,Agrawal et a1.32 demonstrated the decrease 40% -
of the PAF binding to humanblood eosinophilsby 5
0
pretreatmentwith pertussistoxin. Thesefindingssug-
0 NormaiS
gest that there may be a close relationshipbetween
PAF receptorsand or,-adrenergicreceptors(and G i 4 :I- l Asthmatics
0
protein), althoughthe role of guaninenucleotidesin
10% -
PAF-receptorregulationremainsunclear.Additional
studieswill be neededto clarify the mechanismsof 0% 1 -- -..---I--
interaction between a,-adrenoceptorsand PAF re- 0 01 0.1 !

ceptors.
In summary,we demonstrated that m idaglizole, a
specific a,-antagonist, enhancedisoproterenol-in-
ducedCAMPproductionin humanMNCs from normal
subjectsand subjects with asthma,althoughit did not
increasethe CAMP productionby itself. This syner-
gistic effect of m idaglizoleresultedin a higherpercent
increasein subjectswith asthmathan in normal sub-
jects. M idaglizolealsopreventedPAF-induceddesen-
Coflosntratbfl of PAF I
FIG. 5. Effect of PAF on isoproterenol-induced CAMP re-
sponse in MNCs from normal subjects (pi = 71 and sub-
jects with asthma (N = 6). CAMP content in&toed by
1 kmol/L of isoproterenol was taken as a lOO%, and all
other values were calculated as a percent of this CAMP
content; *p < 0.05, compared with control hsoproterenol
alone) by Wilcoxon’s test. No significant differences on
response to PAF between two groups.
728 Sigiyama et al. J. ALLERGY CLIN. IMMUNOL.
M A R C H 1992

TABLE II. Synergistic effect of midaglizole on epinephrine- and isoproterenol-induced CAMP

production in MNCs from subjects with asthma

C A M P production (pMIlO@ MNCs)

lsoproterenol Epinephrine
Conditions (1 pmol/L, N = 10) (1 pmol/L, N = 8)

Stimulant alone 4.0 2 0.6 5.0 5 1.4

M id (10 p,mol/L)plus stimulant 7.2 r l.l* 8.9 -+ 2.2t
% Increase 183.8% ? 18.9% 189.0% r 15.9%

Mid, Midaglizole.
Value of stimulant-inducedCAMP production was obtained by subtractionof a basal CAMP level.
No significant differencesbetweenthe two stimulants by analysis of variancetest.
Comparedwith the value induced by stimulant alone in the samegroup by Wilcoxon’s test:
*p < 0.01.
tp < 0.02.

TABLE III. Effect of midaglizole on PAF-induced desensitization of CAMP response to isoproterenol

in MNCs from normal subjects and subjects with asthma

c A M P content (% control)

Subjects

Normal With asthma

Conditions (N = 7) (N = 8)

PAF (1 kmol/L) 63.3 k 7.0 66.4 k 8.9

PAF plus M id 10 pmol/L* 96.0 f 11.8 t118.1 -c 5.2t
PAF plus Mid 100 p.mol/L* 133.0 +- 12.9f t145.0 ? 9.2$

CAMP content induced by 1 p,mol/L of isoproterenolwas taken as a lOO%, and all other values were calculatedas a percentof it.
*p < 042.
tp < 0.05.
$p < 0.05, comparedwith the control (isoproterenolalone) by Wilcoxon’s test. There were no significant differences betweenthe two
groups by analysis of variance test.

sitization of the CAMP responseto isoproterenolin
MNCs. Thesefindings suggestthat m idaglizolemay
be a useful additional agent in BA through an en-
hancementof the CAMP production.
We thank Mrs. RosemaryBans and M iss NanetteA.
Royle for their help with the preparation of this manuscript.
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NUMBER 3
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