NaunymSchmiedeberg's
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 27-32 (1976)
Pharmacology
9 by Springer-Verlag 1976
Effects of Methacholine, Histamine and Atropine on Pulmonary Guanosine-3',5'-Monophosphate Levels in Hypersensitive Mice JAMES B. POLSON, JOSEPH J. KRZANOWSKI, and A N D O R SZENTIVANYI Department of Pharmacology, University of South Florida College of Medicine, Tampa, Florida 33620, U.S.A.
Summary. Pulmonary levels of cGMP and cAMP in mice sensitized to methacholine and histamine with B. pertussis were examined to determine whether sensitization could be the result of an alteration in the metabolism of these cyclic nucleotides. The results presented show that in sensitized mice, methacholine raised cGMP to levels that were about double those produced without sensitization. In analogous experiments, histamine raised cGMP by approximately 100% in sensitized mice without producing significant increases in nonsensitized groups. Atropine completely blocked the cGMP rises produced by methacholine but did not eliminate those produced by histamine, thus indicating that cholinergic, but not the histaminergic elevation of cGMP involves activation of muscarinic receptors. The influence of pertussis on cAMP appeared to be opposite in direction from cGMP, i.e., a small but significant drop in cAMP levels was found following methacholine administration to sensitized, but not to nonsensitized mice. It was concluded that pertussis sensitization increases the responsiveness of the pulmonary guanylate cyclase-cGMP system to methacholine and histamine, and that the altered patterns of cGMP accumulation may contribute to the biochemical mechanism of sensitization. Key words." Adenosine cyclic 3',5'-monophosphate Guanosine cyclic 3',5'-monophosphate - Bordetella pertussis- Methacholine - Histamine.
INTRODUCTION
Bordetellapertussis organisms, or components thereof, induce potent biological effects in certain strains of Send offprint request to. J. B. Poison at the above address
mice and rats. Among these effects is a heightened susceptibility to histamine, methacholine, 5-hydroxytryptamine, bradykinin and a variety of immunological and physical stimuli (Munoz and Bergman, 1968). This sensitization is accompanied by a decrease in physiological and metabolic responses to epinephrine, indicating that pertussis blocks or reduces beta adrenergic activation (Fishel and Szentivanyi, 1963; Szentivanyi et al., 1963; Gulbenkian et al., 1968). It was suggested (Szentivanyi, 1968) that sensitization and the other biological effects of pertussis may derive from the altered adrenergic responsiveness, and this view is strongly supported by findings that hypersensitivity similar to pertussis-sensitization can be produced by pharmacological beta adrenergic blockade (Fishel et al., 1962, 1968; Bergman and Munoz, 1971; Martorana et al., 1973). Because of the known close association between beta adrenergic receptors and the adenylate cyclasecyclic 3',5'-adenosine monophosphate (cAMP) system (Robinson et al., 1967), a reduction in responsiveness of adenylate cyclase to adrenergic stimulation was considered a likely biochemical correlate of pertussis sensitization (Szentivanyi, 1968). Indeed, reduced adenylate cyclase responsiveness has now been demonstrated in spleens of sensitized mice (Ortez et al., 1975) and in human and murine lymphocytes incubated with the pertussis sensitizing factor (Parker and Morse, 1973). In contrast, studies on lungs obtained from sensitized mice did not show similar reduction in responsiveness to isoproterenol or epinephrine (Krzanowski et al., 1974, 1975, 1976). Further investigation of pulmonary tissue, which is the subject of this report, revealed a possible link between cyclic 3',5'-guanosine monophosphate (cGMP) metabolism and pertussis sensitization as demonstrated by an increase in cGMP levels following administration of methacholine and histamine to sensitized mice. Furthermore, slightly decreased cAMP levels were found
28
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) 900
to a c c o m p a n y t h e p e a k c G M P rise p r o d u c e d b y m e t h a c h o l i n e in sensitized, b u t n o t in n o n s e n s i t i z e d mice, indicating that the pertussis-induced alteration m a y affect b o t h cyclic n u c l e o t i d e s . P r e l i m i n a r y rep o r t s r e l a t e d to this i n v e s t i g a t i o n h a v e b e e n p r e s e n t e d e l s e w h e r e ( P o l s o n et al., 1974a, b, c, 1975).
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MATERIALS
AND METHODS
Sensitization. A total of 313 female mice of the CFW strain (Carworth Farms) and 67 of the ICR strain (Flow Labs) were employed in this investigation. Hypersensitivity was produced by intraperitoneal (i.p.) injection of approximately 7.2x 10 9 heat killed Bordetella pertussis organisms in 0.35 ml volumes of 0.9 ~ NaC1. Five days late1:, which corresponds to the peak development of hypersensitivity (Munoz and Bergman, 1968), 8 - 1 0 randomly selected mice were tested by i.p. administration of histamine (70 mg/ kg) or methacholine (20 mg/kg), using 50 ~ or greater incidence of death within 45 rain as the criterion for sensitization. Other sensitized mice from the same group were used the same day for biochemical studies.
Drug Administration and Tissue Sampling. Histamine dihydrochloride (70 mg/kg), methacholine chloride (5 mg/kg) and atropine sulfate (10 mg/kg) purchased from Sigma Chemical Co. were each administered as free base, i.p. in 0.2ml volumes of 0.9~ NaC1. Time intervals were adopted to correspond to the peak cAMP response to histamine as established in our laboratory (Krzanowski et al., 1974, 1975, 1976), and basal (0-time) conditions were determined without drug administration. Mice were decapitated with scissors held so that upper thoraces were also sheared. The lungs were rapidly removed and plunged into liquid nitrogen within 1.5- 2 s after sacrifice, then stored frozen ( - 9 0 ~C) until assayed.
Tissue Analysis. Frozen samples were dissected and weighed in a Harris cyrostat (-40~ Nucleoside phosphates were extracted from 100-120 mg of tissue into 0.5 ml of 0.5 M perchloric acid (-4~ in Broeck tissue grinders and the protein-free extracts titrated to pH 7 with 2 M K2CO3. The cAMP content of neutralized extracts was measured in triplicate aliquots by protein-binding methodology (Gilman, 1970) and cGMP was measured in duplicate by radioimmunoassay (Steiner et al., 1972) using commercial antiserum (Schwarz/Mann). Cross reactivity of binding reagents was checked to insure that tissue levels of cAMP or cGMP, adenosine, guanosine, AMP, GMP, ADP, GDP, ATP, and GTP did not interfere with assays. The presence of tissue extracts did not influence the assays as determined by measurement of known standards. Also, tissue levels of cGMP measured without column purification of samples were essentially the same as found after purification on Bio-Rad AG 1-X8 resin with formic acid elution (Polson et al., 1974c). Statistics. Comparisons between groups were made using Student's t-test with P < 0.05 adopted as indicative of significant change.
RESULTS
Effect o f Sensitization on Basal c G M P Levels. I n t h e e x p e r i m e n t s u m m a r i z e d in F i g u r e 1, r e s t i n g levels o f c G M P w e r e 5 4 ~ g r e a t e r ( P < 0.05) in s e n s i t i z e d t h a n n o n s e n s i t i z e d m i c e . E l e v a t e d b a s a l c G M P levels a p p e a r to b e a n o c c a s i o n a l , b u t n o t o b l i g a t o r y , at-
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Fig. 1. Effects of methacholine on pulmonary cGMP Ievels in non-sensitized and sensitized CFW strain mice. Tissues were collected without drug administration (0-time) and 5 and 10 rain after methacholine (5 mg/kg, i.p.). The cGMP in each sample was determined by radioimmunoassay. Bars represent means (_+ standard errors) derived from the number of mice shown in parentheses, and asterisks indicate groups significantly different from controI (nonsensitized, 0-time) as follows: * P < 0.05, ** P < 0.001. Elevations found in sensitized mice were statistically different from nonsensitized groups (P < 0.001 at 5 and P < 0.005 at 10 rain)
t r i b u t e o f s e n s i t i z a t i o n b u t this d e g r e e o f d i f f e r e n c e w a s e x c e p t i o n a l , c G M P h a s n e v e r b e e n o b s e r v e d to d e c r e a s e d u e to s e n s i t i z a t i o n . I n c o n t r a s t to c G M P , s e n s i t i z a t i o n d i d n o t s i g n i f i c a n t l y c h a n g e b a s a l levels o f c A M P (Fig. 2).
Effect o f Sensitization on Methacholine-Induced c G M P and c A M P Responses. A t 5 a n d 10 m i n a f t e r m e t h a c h o l i n e (5 m g / k g ) , t h e levels o f c G M P w e r e r a i s e d in n o n s e n s i t i z e d m i c e t o 7 a n d 2 t i m e s c o n t r o l , res p e c t i v e l y , a n d in s e n s i t i z e d m i c e to 12 a n d 4 t i m e s
J. B. Poison et al. : Pulmonary cGMP in Hypersensitive Mice
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Fig. 2. Pulmonary levels of cAMP in nonsensitized and sensitized CFW strain mice following administration of methacholine. Tissues were collected without drug administration (0-time) and 5 and 10 rain after methacholine (5 mg/kg, i.p.). The cAMP in each sample was determined by protein-binding methodology. Bars represent means (+ standard errors) derived from the number of mice shown in parentheses. No mean was statistically different from control (nonsensitized, 0-time); however, significant differenceswere found between nonsensitized versus sensitized mice at 5 rain (P < 0.005) and between sensitized groups as follows: 0 versus 5 rain, P < 0.02; 5 versus I0 rain, P < 0.05
control (Fig9 1); changes that were significantly greater than the increases observed without sensitization (P < 0.001 at 5; P < 0.05 at 10 rain). Changes in the levels of c A M P produced by methacholine were relatively small (Fig. 2), none being statistically different f r o m the control (nonsensitized, no methacholine) group. Levels of c A M P were not significantly modified by sensitization; however, after sensitization, methacholine significantly lowered c A M P by 1 6 ~ (P < 0.02) by 5 min, levels having returned to control by 10 rain.
Effect of Atropine on Methacholine-Indueed cGMP Responses. In order to investigate the pharmacological specificity o f c G M P changes, atropine was selected as a specific muscarinic blocking agent. This agent alone caused c G M P to fall 33 and 38 ~ 15 rain after its administration to sensitized and nonsensitized mice, respectively (Fig. 3). This drop was statistically significant (P < 0.05) for nonsensitized mice, but because of large variation, it was not statistically significant for the sensitized group in this series of animals.
CONTRQL
ATROPINE
METHACHOLINE COMBINATION
Fig. 3. Influence of atropine on cGMP rises produced by methacholine in CFW strain mice. Atropine (10 mg/kg, i.p.) was administered 10 rain before methacholine (5 mg/kg, i.p.) and 15 rain before collection of samples. Control groups received no drug. Bars represent means (+ standard errors) derived from the number of mice shown in parentheses, and asterisks indicate groups significantly different from nonsensitized controls as follows: *P < 0.05, **P < 0.001. Elevations produced by methacholine in the absence of atropine were significantly different from all other groups (P < 0.001)
However, sensitized mice shown in Figure 4 receiving the same dosage of atropine responded with somewhat less variation and the drop (30 ~ ) in c G M P , qualitatively and quantitatively the same, was significant by statistical criteria (P < 0.05). Administration of atropine 10 rain before methacholine completely blocked rises in c G M P in b o t h nonsensitized and sensitized mice (Fig. 3), demonstrating that these rises are dependent on the stimulation of muscarinic receptors.
Effect of Blocking Agents" on Histamine-Induced cGMP Responses. Histamine has also been found to elevate p u l m o n a r y c G M P in sensitized, but not in nonsensitized I C R strain mice (Polson et al., 1974c). Based on the information presented in Figure 1 and 3, it seemed conceivable that these elevations could have resulted from histamine-induced release of acetylcholine f r o m endogenous sites9 In order to investigate this possibility, atropine (10 mg/kg) was administered to sensitized mice 10 min before histamine (70 mg/kg) under the same conditions that blocked methacholine-
30
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) 100
l-I
WITHOUT HISTAMINE
HISTAMINE
"% ~r
Table 1. Influence ofdiphenhydramine and burimamide on cGMP rises produced by histamine in pertussis-sensitized ICR strain mice. Diphenhydramine and burimamide were administered at
dose levels of 25 mg/kg (i.p.) 5 min before collection of pulmonary tissue, cGMP levels were determined by radioimmunoassay. Each percent increase reported is the difference between cGMP levels in non-histamine treated (100~ baseline) and histamine treated mice shown in the same column. P values were calculated by Student's t-test
+1
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Fig. 4. Elevation of cGMP by histamine in pulmonary tissues of sensitized CFW strain mice with and without prior administration
of atropine. The muscarinic blocking agent (10 mg/kg, i.p.) was administered 10 min before histamine (70 mg/kg, i.p.) and 15 min before collection of samples. Bars represent means (_+ standard errors) derived from the number of mice shown in parentheses. The drop in cGMP levelsproduced by atropine was statisticallysignificant only between groups not given histamine (P < 0.05). Nonsensitized mice used as controls showed no significant elevation after histamine, but a significant drop (P < 0.005) after atropine with cGMP levels +_ S.E. (n) as follows: no drug, 36.7 • 2.7 (8); histamine, 41.5 _+ 3.8 (8); atropine, 26.3 _+ 1.2 (10); both agents, 26.0 _+ 1.8 (10)
induced c G M P rises. The results shown in Figure 4 reveal that, although the response to histamine may have been slightly reduced, a prominent portion of the effect was not blocked. In contrast, the antihistaminic agent diphenhydramine, which blocks H1 receptors (Ash and Schild, 1966), was found to produce effective blockade of the histamine-induced c G M P rise (Table 1). Burimamide, which is more specific for H2 receptors (Black et al., 1972), did not block the c G M P rise as effectively as diphenhydramine, despite the fact that the 25 mg/kg dose levels used for both antihistaminic agents provided about 29 ~ more burimamide on a mole per mole basis due to burimamide's lower molecular weight (198 versus 255). DISCUSSION The data presented indicate that pertussis-induced sensitization to pharmacological agents results in increased p u l m o n a r y accumulations of c G M P in response to methacholine and histamine. Atropine was selected as a muscarinic blocking agent to examine
Control
Diphenhydramine
Burimamide
63.4 • 5.6 (9) 116.8 • 20.5 (S)
48.5 _+4.5 (10) 55.7 _+4.3 (10) *
56.1 • 3.8 (10) 86.8 _+6.8 (10)
Percent increase 84 P value** < 0.05
15 55 not significant < 0.005
* Significantly different from other histamine treated groups (P < 0.01) ** Comparison between histamine treated and non-histamine treated mice in same column
the pharmacological specificity of these c G M P accumulations. This agent completely eliminated the elevation of c G M P by methacholine in both sensitized and nonsensitized lung, demonstrating the importance of muscarinic receptors in this cholinergic response. Furthermore, atropine alone decreased c G M P , suggesting that basal levels were maintained by cholinergic nerve activity in the intact animals. These findings strengthen the inference that autonomic effector (i.e., smooth muscle and/or secretory) cells are involved in the c G M P response to cholinergic activation by methacholine. Since the elevation of c G M P by histamine was not blocked by atropine, it seems unlikely that the major part of the histamine-induced c G M P rise could have resulted from a release of acetylcholine from endogenous sites or that sensitization by B. pertussis can be explained by an increase in cholinergic neuronal activity. Furthermore, since the effect of histamine was blocked by diphenhydramine, but much less effectively blocked by burimamide, it appears that the elevation of c G M P levels by histamine is dependent on stimulation of H t receptors. Since sensitization has been shown to be accompanied by an increased tolerance of mice and rats to beta adrenergic agents (Szentivanyi et al., 1963), it was suggested (Szentivanyi, 1968) that pharmacological hypersensitivity and other biological effects of pertussis m a y derive from this reduced adrenergic responsiveness. This view is strongly supported by
J. B. Polson et al. : Pulmonary cGMP in Hypersensitive Mice
findings that pharmacological beta adrenergic blocking agents can produce sensitivity resembling pertussis sensitization (Fishel et al., 1963, 1968; Bergman and Munoz, 1971; Martorana et al., 1973). In this context, the results showing a slight, but significant decrease in cAMP (Fig. 2) corresponding with the large rise in cGMP found at 5 min after administration of methacholine to sensitized mice (Fig. 1) may be of potential significance. It is possible that reduced beta adrenergic activity leads to increased accumulation of cGMP in pulmonary cells. Kuo and Kuo (1973) showed that cholinergically elevated cGMP levels in rat lung slices were lowered by isoproterenol, indicating that cGMP accumulation was inhibited by beta adrenergic activation. Furthermore, it has been shown that beta adrenergic blockade with propranolol increases the susceptibility of pulmonary cGMP to elevation by histamine (Polson et al., 1974c). However, further information is required before conclusions can be drawn as to whether the primary abnormality produced by pertussis is on some element of beta adrenergic mechanisms, directly on guanylate cyclase, on cyclic nucleotide phosphodiesterases, or on some other possible site of action. In addition to its pharmacological implications, pertussis sensitization is a useful laboratory model for the study of human atopic disease because it reproduces the atopic abnormality (Szentivanyi, 1968; Reed, 1968). In this context, abnormal cyclic nucleotide responsiveness similar to that found after sensitization is implicated in the pathogenesis of bronchial asthma and related diseases (Polson et al., 1974a, c, 1975; Krzanowski et al., 1976). Acknowledgements. The authors thank Dolly Hwang, Constance McCleary, Mary Moffler and Jean Parks for excellent technical assistance. The investigation was supported by grants from National Institutes of Health IR23HL17106 and the National Science Foundation GB-35244.
REFERENCES Ash, A. S. F., Schild, H. O. : Receptors mediating some actions of histamine. Brit. J. Pharmacol. 27, 427-439 (1966) Bergman, R. K., Munoz, J. : Effects of epinephrine, norepinephrine and isoproterenoI agonist challenge in BordeteIla pertussistreated and beta-adrenergic blocked mice. Life Sci. 10, 561 - 568 (1971) Black, J.W., Duncan, W. A. M., Durant, C. J., Ganellin, C. R., Parsons, E. M.: Definition and antagonism of histamine H2receptors. Nature (Lond.) 236, 385-390 (1972) Fishel, C. W., Keller, K. F., O'Bryan, B. S. : Toxicity of combined histamine and serotonin in normal, pertussis-sensitized, and fl-adrenergically blocked mice. J. Immunol. 101, 679-685 (1968) Fishel, C. W., Szentivanyi, A. : The absence of adrenaline-induced hyperglycemia in pertussis-sensitized mice and its relation to histamine and serotonin hypersensitivity. J. Allergy 34, 439-454 (1963)
31 Fishel, C. W., Szentivanyi, A., Talmage, D. W. : Sensitization and desensitization of mice to histamine and serotonin by neurohumors. J. Immunol. 89, 8 - 1 8 (1962) Gilman, A. G. : A protein binding assay for adenosine-3',5'-cyclic monophosphate. Proc. nat. Acad. Sci. (Wash.) 67, 305-312 (1970) Gulbenkian, A., Schobert, L., Nixon, C., Tabachnick, I. I. A. : Metabolic effects of pertussis sensitization in mice and rats. Endocrinology 83, 885-892 (1968) Krzanowski, J., Polson, J., Szentivanyi, A. : Histamine, isoproterenol, and epinephrine induced in vivo activation of adenyl cyclase in mouse lung following B. pertussis. Pharmacologist 16, 212 (1974) Krzanowski, J. J., Jr., Polson, J. B., Szentivanyi, A. : In vivo alterations in adenosine-3',5'-cyclic monophosphate levels following adrenergic activation in an animal model of atopic disease. In: Advances in cyclic nucleotide research, Vol. 5 (G. I. Drummond, P. Greengard, and G. A. Robison, eds.), p. 815. New York: Raven Press 1975 Krzanowski, J. J., Polson, J. B., Szentivanyi, A.: Pulmonary patterns of adenosine-3',5'-cyclic monophosphate accumulations in response to adrenergic or histamine stimulation in Bordetella pertussis sensitized mice. In press: Biochem. Pharmacol. 25, (1976) Kuo, J. F., Kuo, W. N. : Regulation by fl-adrenergic receptor and muscarinic cholinergic receptor activation of intracellular cyclic AMP and cyclic GMP levels in rat lung slices. Biochem. biophys. Res. Commun. 55, 660-665 (1973) Martorana, P. A., Richard, J. W., Share, N. N. : Difference between B. pertussis and propranolol induced histamine hypersensitivity in mice. Canad. J. Physiol. Pharmacol. 51, 102-107 (1973) Munoz, J., Bergman, R. K.: Histamine-sensitizing factors from microbial agents with special reference to Bordetella pertussis. Bact. Rev. 32, 103-126 (1968) Ortez, R. A., Seshachalam, D., Szentivanyi, A. : Alterations in adenyI cyclase activity and glucose utilization of Bordetella pertussis-sensitized mouse spleen. Biochem. Pharmacol. 24, 1297-1302 (1975) Parker, C. W., Morse, S. I. : The effect of Bordetella pertussis on lymphocyte cyclic AMP metabolism. J. exp. Med. 137, 10781090 (1973) Poison, J. B., Krzanowski, J. J., Szentivanyi, A. : Effect of histamine on the pulmonary levels of cyclic nucleotides in normal mice and under conditions of pharmacologic or bacterial sensitization. J. Allergy clin. Immunol. 53, 100 (1974a) Poison, J. B., Krzanowski, J. J., Szentivanyi, A. : Effects of methacholine, histamine, and atropine on pulmonary guanosine-3',5'cyclic monophosphate in hypersensitive mice. Pharmacologist 16, 212 (1974b) Polson, J. B., Krzanowski, J.J., Szentivanyi, A.: Histamine induced changes in pulmonary guanosine-3',5'-cyclic monophosphate and adenosine-3',5'-cyclic monophosphate levels in mice following sensitization by Bordetella pertussis and/or propranolol. Res. Commun. chem. Path. Pharmacol. 9, 243-251 (1974c) Polson, J. B., Krzanowski, J. J., Jr., Szentivanyi, A.: Elevation of pulmonary cyclic GMP levels caused by histamine and methacholine in mice simulating atopic disease. In: Advances in cyclic nucleotide research, Vol. 5 (G. I. Drummond, P. Greengard, and G. A. Robison, eds.), p. 815. New York: Raven Press 1975 Reed, C. E. : Pertussis sensitization as an animal model for the abnormal bronchial sensitivity of asthma. Yale J. Biol. Med. 40, 507-515 (1968) Robison, G. A., Butcher, R. W., Sutherland, E. W. : Adenyl cyclase as an adrenergic receptor. Ann. N. Y. Acad. Sci. 139, 703 - 723 (1967)
32 Steiner, A. L., Parker, C. W., Kipnis, D. M. : Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. biol. Chem. 247, 1106-1113 (1972) Szentivanyi, A. : The beta adrenergic theory of the atopic abnormality in bronchial asthma. J. Allergy clin. Immunol. 42, 203 - 232 (1968)
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) Szentivanyi, A., Fishel, C. W., Talmage, D. W. : Adrenaline mediation of histamine and serotonin hyperglycemia in normal mice and the absence of adrenaline-induced hyperglycemia in pertussis-sensitized mice. J. infect. Dis. 113, 86-98 (1963)
Received February 11 / Accepted May 31, 1976
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 27-32 (1976)
Pharmacology
9 by Springer-Verlag 1976
Effects of Methacholine, Histamine and Atropine on Pulmonary Guanosine-3',5'-Monophosphate Levels in Hypersensitive Mice JAMES B. POLSON, JOSEPH J. KRZANOWSKI, and A N D O R SZENTIVANYI Department of Pharmacology, University of South Florida College of Medicine, Tampa, Florida 33620, U.S.A.
Summary. Pulmonary levels of cGMP and cAMP in mice sensitized to methacholine and histamine with B. pertussis were examined to determine whether sensitization could be the result of an alteration in the metabolism of these cyclic nucleotides. The results presented show that in sensitized mice, methacholine raised cGMP to levels that were about double those produced without sensitization. In analogous experiments, histamine raised cGMP by approximately 100% in sensitized mice without producing significant increases in nonsensitized groups. Atropine completely blocked the cGMP rises produced by methacholine but did not eliminate those produced by histamine, thus indicating that cholinergic, but not the histaminergic elevation of cGMP involves activation of muscarinic receptors. The influence of pertussis on cAMP appeared to be opposite in direction from cGMP, i.e., a small but significant drop in cAMP levels was found following methacholine administration to sensitized, but not to nonsensitized mice. It was concluded that pertussis sensitization increases the responsiveness of the pulmonary guanylate cyclase-cGMP system to methacholine and histamine, and that the altered patterns of cGMP accumulation may contribute to the biochemical mechanism of sensitization. Key words." Adenosine cyclic 3',5'-monophosphate Guanosine cyclic 3',5'-monophosphate - Bordetella pertussis- Methacholine - Histamine.
INTRODUCTION
Bordetellapertussis organisms, or components thereof, induce potent biological effects in certain strains of Send offprint request to. J. B. Poison at the above address
mice and rats. Among these effects is a heightened susceptibility to histamine, methacholine, 5-hydroxytryptamine, bradykinin and a variety of immunological and physical stimuli (Munoz and Bergman, 1968). This sensitization is accompanied by a decrease in physiological and metabolic responses to epinephrine, indicating that pertussis blocks or reduces beta adrenergic activation (Fishel and Szentivanyi, 1963; Szentivanyi et al., 1963; Gulbenkian et al., 1968). It was suggested (Szentivanyi, 1968) that sensitization and the other biological effects of pertussis may derive from the altered adrenergic responsiveness, and this view is strongly supported by findings that hypersensitivity similar to pertussis-sensitization can be produced by pharmacological beta adrenergic blockade (Fishel et al., 1962, 1968; Bergman and Munoz, 1971; Martorana et al., 1973). Because of the known close association between beta adrenergic receptors and the adenylate cyclasecyclic 3',5'-adenosine monophosphate (cAMP) system (Robinson et al., 1967), a reduction in responsiveness of adenylate cyclase to adrenergic stimulation was considered a likely biochemical correlate of pertussis sensitization (Szentivanyi, 1968). Indeed, reduced adenylate cyclase responsiveness has now been demonstrated in spleens of sensitized mice (Ortez et al., 1975) and in human and murine lymphocytes incubated with the pertussis sensitizing factor (Parker and Morse, 1973). In contrast, studies on lungs obtained from sensitized mice did not show similar reduction in responsiveness to isoproterenol or epinephrine (Krzanowski et al., 1974, 1975, 1976). Further investigation of pulmonary tissue, which is the subject of this report, revealed a possible link between cyclic 3',5'-guanosine monophosphate (cGMP) metabolism and pertussis sensitization as demonstrated by an increase in cGMP levels following administration of methacholine and histamine to sensitized mice. Furthermore, slightly decreased cAMP levels were found
28
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) 900
to a c c o m p a n y t h e p e a k c G M P rise p r o d u c e d b y m e t h a c h o l i n e in sensitized, b u t n o t in n o n s e n s i t i z e d mice, indicating that the pertussis-induced alteration m a y affect b o t h cyclic n u c l e o t i d e s . P r e l i m i n a r y rep o r t s r e l a t e d to this i n v e s t i g a t i o n h a v e b e e n p r e s e n t e d e l s e w h e r e ( P o l s o n et al., 1974a, b, c, 1975).
T
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- ~ :~
700
NONSENSITIZED
\\\ "
MATERIALS
AND METHODS
Sensitization. A total of 313 female mice of the CFW strain (Carworth Farms) and 67 of the ICR strain (Flow Labs) were employed in this investigation. Hypersensitivity was produced by intraperitoneal (i.p.) injection of approximately 7.2x 10 9 heat killed Bordetella pertussis organisms in 0.35 ml volumes of 0.9 ~ NaC1. Five days late1:, which corresponds to the peak development of hypersensitivity (Munoz and Bergman, 1968), 8 - 1 0 randomly selected mice were tested by i.p. administration of histamine (70 mg/ kg) or methacholine (20 mg/kg), using 50 ~ or greater incidence of death within 45 rain as the criterion for sensitization. Other sensitized mice from the same group were used the same day for biochemical studies.
Drug Administration and Tissue Sampling. Histamine dihydrochloride (70 mg/kg), methacholine chloride (5 mg/kg) and atropine sulfate (10 mg/kg) purchased from Sigma Chemical Co. were each administered as free base, i.p. in 0.2ml volumes of 0.9~ NaC1. Time intervals were adopted to correspond to the peak cAMP response to histamine as established in our laboratory (Krzanowski et al., 1974, 1975, 1976), and basal (0-time) conditions were determined without drug administration. Mice were decapitated with scissors held so that upper thoraces were also sheared. The lungs were rapidly removed and plunged into liquid nitrogen within 1.5- 2 s after sacrifice, then stored frozen ( - 9 0 ~C) until assayed.
Tissue Analysis. Frozen samples were dissected and weighed in a Harris cyrostat (-40~ Nucleoside phosphates were extracted from 100-120 mg of tissue into 0.5 ml of 0.5 M perchloric acid (-4~ in Broeck tissue grinders and the protein-free extracts titrated to pH 7 with 2 M K2CO3. The cAMP content of neutralized extracts was measured in triplicate aliquots by protein-binding methodology (Gilman, 1970) and cGMP was measured in duplicate by radioimmunoassay (Steiner et al., 1972) using commercial antiserum (Schwarz/Mann). Cross reactivity of binding reagents was checked to insure that tissue levels of cAMP or cGMP, adenosine, guanosine, AMP, GMP, ADP, GDP, ATP, and GTP did not interfere with assays. The presence of tissue extracts did not influence the assays as determined by measurement of known standards. Also, tissue levels of cGMP measured without column purification of samples were essentially the same as found after purification on Bio-Rad AG 1-X8 resin with formic acid elution (Polson et al., 1974c). Statistics. Comparisons between groups were made using Student's t-test with P < 0.05 adopted as indicative of significant change.
RESULTS
Effect o f Sensitization on Basal c G M P Levels. I n t h e e x p e r i m e n t s u m m a r i z e d in F i g u r e 1, r e s t i n g levels o f c G M P w e r e 5 4 ~ g r e a t e r ( P < 0.05) in s e n s i t i z e d t h a n n o n s e n s i t i z e d m i c e . E l e v a t e d b a s a l c G M P levels a p p e a r to b e a n o c c a s i o n a l , b u t n o t o b l i g a t o r y , at-
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Fig. 1. Effects of methacholine on pulmonary cGMP Ievels in non-sensitized and sensitized CFW strain mice. Tissues were collected without drug administration (0-time) and 5 and 10 rain after methacholine (5 mg/kg, i.p.). The cGMP in each sample was determined by radioimmunoassay. Bars represent means (_+ standard errors) derived from the number of mice shown in parentheses, and asterisks indicate groups significantly different from controI (nonsensitized, 0-time) as follows: * P < 0.05, ** P < 0.001. Elevations found in sensitized mice were statistically different from nonsensitized groups (P < 0.001 at 5 and P < 0.005 at 10 rain)
t r i b u t e o f s e n s i t i z a t i o n b u t this d e g r e e o f d i f f e r e n c e w a s e x c e p t i o n a l , c G M P h a s n e v e r b e e n o b s e r v e d to d e c r e a s e d u e to s e n s i t i z a t i o n . I n c o n t r a s t to c G M P , s e n s i t i z a t i o n d i d n o t s i g n i f i c a n t l y c h a n g e b a s a l levels o f c A M P (Fig. 2).
Effect o f Sensitization on Methacholine-Induced c G M P and c A M P Responses. A t 5 a n d 10 m i n a f t e r m e t h a c h o l i n e (5 m g / k g ) , t h e levels o f c G M P w e r e r a i s e d in n o n s e n s i t i z e d m i c e t o 7 a n d 2 t i m e s c o n t r o l , res p e c t i v e l y , a n d in s e n s i t i z e d m i c e to 12 a n d 4 t i m e s
J. B. Poison et al. : Pulmonary cGMP in Hypersensitive Mice
29 700
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Fig. 2. Pulmonary levels of cAMP in nonsensitized and sensitized CFW strain mice following administration of methacholine. Tissues were collected without drug administration (0-time) and 5 and 10 rain after methacholine (5 mg/kg, i.p.). The cAMP in each sample was determined by protein-binding methodology. Bars represent means (+ standard errors) derived from the number of mice shown in parentheses. No mean was statistically different from control (nonsensitized, 0-time); however, significant differenceswere found between nonsensitized versus sensitized mice at 5 rain (P < 0.005) and between sensitized groups as follows: 0 versus 5 rain, P < 0.02; 5 versus I0 rain, P < 0.05
control (Fig9 1); changes that were significantly greater than the increases observed without sensitization (P < 0.001 at 5; P < 0.05 at 10 rain). Changes in the levels of c A M P produced by methacholine were relatively small (Fig. 2), none being statistically different f r o m the control (nonsensitized, no methacholine) group. Levels of c A M P were not significantly modified by sensitization; however, after sensitization, methacholine significantly lowered c A M P by 1 6 ~ (P < 0.02) by 5 min, levels having returned to control by 10 rain.
Effect of Atropine on Methacholine-Indueed cGMP Responses. In order to investigate the pharmacological specificity o f c G M P changes, atropine was selected as a specific muscarinic blocking agent. This agent alone caused c G M P to fall 33 and 38 ~ 15 rain after its administration to sensitized and nonsensitized mice, respectively (Fig. 3). This drop was statistically significant (P < 0.05) for nonsensitized mice, but because of large variation, it was not statistically significant for the sensitized group in this series of animals.
CONTRQL
ATROPINE
METHACHOLINE COMBINATION
Fig. 3. Influence of atropine on cGMP rises produced by methacholine in CFW strain mice. Atropine (10 mg/kg, i.p.) was administered 10 rain before methacholine (5 mg/kg, i.p.) and 15 rain before collection of samples. Control groups received no drug. Bars represent means (+ standard errors) derived from the number of mice shown in parentheses, and asterisks indicate groups significantly different from nonsensitized controls as follows: *P < 0.05, **P < 0.001. Elevations produced by methacholine in the absence of atropine were significantly different from all other groups (P < 0.001)
However, sensitized mice shown in Figure 4 receiving the same dosage of atropine responded with somewhat less variation and the drop (30 ~ ) in c G M P , qualitatively and quantitatively the same, was significant by statistical criteria (P < 0.05). Administration of atropine 10 rain before methacholine completely blocked rises in c G M P in b o t h nonsensitized and sensitized mice (Fig. 3), demonstrating that these rises are dependent on the stimulation of muscarinic receptors.
Effect of Blocking Agents" on Histamine-Induced cGMP Responses. Histamine has also been found to elevate p u l m o n a r y c G M P in sensitized, but not in nonsensitized I C R strain mice (Polson et al., 1974c). Based on the information presented in Figure 1 and 3, it seemed conceivable that these elevations could have resulted from histamine-induced release of acetylcholine f r o m endogenous sites9 In order to investigate this possibility, atropine (10 mg/kg) was administered to sensitized mice 10 min before histamine (70 mg/kg) under the same conditions that blocked methacholine-
30
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) 100
l-I
WITHOUT HISTAMINE
HISTAMINE
"% ~r
Table 1. Influence ofdiphenhydramine and burimamide on cGMP rises produced by histamine in pertussis-sensitized ICR strain mice. Diphenhydramine and burimamide were administered at
dose levels of 25 mg/kg (i.p.) 5 min before collection of pulmonary tissue, cGMP levels were determined by radioimmunoassay. Each percent increase reported is the difference between cGMP levels in non-histamine treated (100~ baseline) and histamine treated mice shown in the same column. P values were calculated by Student's t-test
+1
-;
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bJ-: 9
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No histamine
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Histamine 0
181
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NO ATROPINE
i 18) ATROPINE
Fig. 4. Elevation of cGMP by histamine in pulmonary tissues of sensitized CFW strain mice with and without prior administration
of atropine. The muscarinic blocking agent (10 mg/kg, i.p.) was administered 10 min before histamine (70 mg/kg, i.p.) and 15 min before collection of samples. Bars represent means (_+ standard errors) derived from the number of mice shown in parentheses. The drop in cGMP levelsproduced by atropine was statisticallysignificant only between groups not given histamine (P < 0.05). Nonsensitized mice used as controls showed no significant elevation after histamine, but a significant drop (P < 0.005) after atropine with cGMP levels +_ S.E. (n) as follows: no drug, 36.7 • 2.7 (8); histamine, 41.5 _+ 3.8 (8); atropine, 26.3 _+ 1.2 (10); both agents, 26.0 _+ 1.8 (10)
induced c G M P rises. The results shown in Figure 4 reveal that, although the response to histamine may have been slightly reduced, a prominent portion of the effect was not blocked. In contrast, the antihistaminic agent diphenhydramine, which blocks H1 receptors (Ash and Schild, 1966), was found to produce effective blockade of the histamine-induced c G M P rise (Table 1). Burimamide, which is more specific for H2 receptors (Black et al., 1972), did not block the c G M P rise as effectively as diphenhydramine, despite the fact that the 25 mg/kg dose levels used for both antihistaminic agents provided about 29 ~ more burimamide on a mole per mole basis due to burimamide's lower molecular weight (198 versus 255). DISCUSSION The data presented indicate that pertussis-induced sensitization to pharmacological agents results in increased p u l m o n a r y accumulations of c G M P in response to methacholine and histamine. Atropine was selected as a muscarinic blocking agent to examine
Control
Diphenhydramine
Burimamide
63.4 • 5.6 (9) 116.8 • 20.5 (S)
48.5 _+4.5 (10) 55.7 _+4.3 (10) *
56.1 • 3.8 (10) 86.8 _+6.8 (10)
Percent increase 84 P value** < 0.05
15 55 not significant < 0.005
* Significantly different from other histamine treated groups (P < 0.01) ** Comparison between histamine treated and non-histamine treated mice in same column
the pharmacological specificity of these c G M P accumulations. This agent completely eliminated the elevation of c G M P by methacholine in both sensitized and nonsensitized lung, demonstrating the importance of muscarinic receptors in this cholinergic response. Furthermore, atropine alone decreased c G M P , suggesting that basal levels were maintained by cholinergic nerve activity in the intact animals. These findings strengthen the inference that autonomic effector (i.e., smooth muscle and/or secretory) cells are involved in the c G M P response to cholinergic activation by methacholine. Since the elevation of c G M P by histamine was not blocked by atropine, it seems unlikely that the major part of the histamine-induced c G M P rise could have resulted from a release of acetylcholine from endogenous sites or that sensitization by B. pertussis can be explained by an increase in cholinergic neuronal activity. Furthermore, since the effect of histamine was blocked by diphenhydramine, but much less effectively blocked by burimamide, it appears that the elevation of c G M P levels by histamine is dependent on stimulation of H t receptors. Since sensitization has been shown to be accompanied by an increased tolerance of mice and rats to beta adrenergic agents (Szentivanyi et al., 1963), it was suggested (Szentivanyi, 1968) that pharmacological hypersensitivity and other biological effects of pertussis m a y derive from this reduced adrenergic responsiveness. This view is strongly supported by
J. B. Polson et al. : Pulmonary cGMP in Hypersensitive Mice
findings that pharmacological beta adrenergic blocking agents can produce sensitivity resembling pertussis sensitization (Fishel et al., 1963, 1968; Bergman and Munoz, 1971; Martorana et al., 1973). In this context, the results showing a slight, but significant decrease in cAMP (Fig. 2) corresponding with the large rise in cGMP found at 5 min after administration of methacholine to sensitized mice (Fig. 1) may be of potential significance. It is possible that reduced beta adrenergic activity leads to increased accumulation of cGMP in pulmonary cells. Kuo and Kuo (1973) showed that cholinergically elevated cGMP levels in rat lung slices were lowered by isoproterenol, indicating that cGMP accumulation was inhibited by beta adrenergic activation. Furthermore, it has been shown that beta adrenergic blockade with propranolol increases the susceptibility of pulmonary cGMP to elevation by histamine (Polson et al., 1974c). However, further information is required before conclusions can be drawn as to whether the primary abnormality produced by pertussis is on some element of beta adrenergic mechanisms, directly on guanylate cyclase, on cyclic nucleotide phosphodiesterases, or on some other possible site of action. In addition to its pharmacological implications, pertussis sensitization is a useful laboratory model for the study of human atopic disease because it reproduces the atopic abnormality (Szentivanyi, 1968; Reed, 1968). In this context, abnormal cyclic nucleotide responsiveness similar to that found after sensitization is implicated in the pathogenesis of bronchial asthma and related diseases (Polson et al., 1974a, c, 1975; Krzanowski et al., 1976). Acknowledgements. The authors thank Dolly Hwang, Constance McCleary, Mary Moffler and Jean Parks for excellent technical assistance. The investigation was supported by grants from National Institutes of Health IR23HL17106 and the National Science Foundation GB-35244.
REFERENCES Ash, A. S. F., Schild, H. O. : Receptors mediating some actions of histamine. Brit. J. Pharmacol. 27, 427-439 (1966) Bergman, R. K., Munoz, J. : Effects of epinephrine, norepinephrine and isoproterenoI agonist challenge in BordeteIla pertussistreated and beta-adrenergic blocked mice. Life Sci. 10, 561 - 568 (1971) Black, J.W., Duncan, W. A. M., Durant, C. J., Ganellin, C. R., Parsons, E. M.: Definition and antagonism of histamine H2receptors. Nature (Lond.) 236, 385-390 (1972) Fishel, C. W., Keller, K. F., O'Bryan, B. S. : Toxicity of combined histamine and serotonin in normal, pertussis-sensitized, and fl-adrenergically blocked mice. J. Immunol. 101, 679-685 (1968) Fishel, C. W., Szentivanyi, A. : The absence of adrenaline-induced hyperglycemia in pertussis-sensitized mice and its relation to histamine and serotonin hypersensitivity. J. Allergy 34, 439-454 (1963)
31 Fishel, C. W., Szentivanyi, A., Talmage, D. W. : Sensitization and desensitization of mice to histamine and serotonin by neurohumors. J. Immunol. 89, 8 - 1 8 (1962) Gilman, A. G. : A protein binding assay for adenosine-3',5'-cyclic monophosphate. Proc. nat. Acad. Sci. (Wash.) 67, 305-312 (1970) Gulbenkian, A., Schobert, L., Nixon, C., Tabachnick, I. I. A. : Metabolic effects of pertussis sensitization in mice and rats. Endocrinology 83, 885-892 (1968) Krzanowski, J., Polson, J., Szentivanyi, A. : Histamine, isoproterenol, and epinephrine induced in vivo activation of adenyl cyclase in mouse lung following B. pertussis. Pharmacologist 16, 212 (1974) Krzanowski, J. J., Jr., Polson, J. B., Szentivanyi, A. : In vivo alterations in adenosine-3',5'-cyclic monophosphate levels following adrenergic activation in an animal model of atopic disease. In: Advances in cyclic nucleotide research, Vol. 5 (G. I. Drummond, P. Greengard, and G. A. Robison, eds.), p. 815. New York: Raven Press 1975 Krzanowski, J. J., Polson, J. B., Szentivanyi, A.: Pulmonary patterns of adenosine-3',5'-cyclic monophosphate accumulations in response to adrenergic or histamine stimulation in Bordetella pertussis sensitized mice. In press: Biochem. Pharmacol. 25, (1976) Kuo, J. F., Kuo, W. N. : Regulation by fl-adrenergic receptor and muscarinic cholinergic receptor activation of intracellular cyclic AMP and cyclic GMP levels in rat lung slices. Biochem. biophys. Res. Commun. 55, 660-665 (1973) Martorana, P. A., Richard, J. W., Share, N. N. : Difference between B. pertussis and propranolol induced histamine hypersensitivity in mice. Canad. J. Physiol. Pharmacol. 51, 102-107 (1973) Munoz, J., Bergman, R. K.: Histamine-sensitizing factors from microbial agents with special reference to Bordetella pertussis. Bact. Rev. 32, 103-126 (1968) Ortez, R. A., Seshachalam, D., Szentivanyi, A. : Alterations in adenyI cyclase activity and glucose utilization of Bordetella pertussis-sensitized mouse spleen. Biochem. Pharmacol. 24, 1297-1302 (1975) Parker, C. W., Morse, S. I. : The effect of Bordetella pertussis on lymphocyte cyclic AMP metabolism. J. exp. Med. 137, 10781090 (1973) Poison, J. B., Krzanowski, J. J., Szentivanyi, A. : Effect of histamine on the pulmonary levels of cyclic nucleotides in normal mice and under conditions of pharmacologic or bacterial sensitization. J. Allergy clin. Immunol. 53, 100 (1974a) Poison, J. B., Krzanowski, J. J., Szentivanyi, A. : Effects of methacholine, histamine, and atropine on pulmonary guanosine-3',5'cyclic monophosphate in hypersensitive mice. Pharmacologist 16, 212 (1974b) Polson, J. B., Krzanowski, J.J., Szentivanyi, A.: Histamine induced changes in pulmonary guanosine-3',5'-cyclic monophosphate and adenosine-3',5'-cyclic monophosphate levels in mice following sensitization by Bordetella pertussis and/or propranolol. Res. Commun. chem. Path. Pharmacol. 9, 243-251 (1974c) Polson, J. B., Krzanowski, J. J., Jr., Szentivanyi, A.: Elevation of pulmonary cyclic GMP levels caused by histamine and methacholine in mice simulating atopic disease. In: Advances in cyclic nucleotide research, Vol. 5 (G. I. Drummond, P. Greengard, and G. A. Robison, eds.), p. 815. New York: Raven Press 1975 Reed, C. E. : Pertussis sensitization as an animal model for the abnormal bronchial sensitivity of asthma. Yale J. Biol. Med. 40, 507-515 (1968) Robison, G. A., Butcher, R. W., Sutherland, E. W. : Adenyl cyclase as an adrenergic receptor. Ann. N. Y. Acad. Sci. 139, 703 - 723 (1967)
32 Steiner, A. L., Parker, C. W., Kipnis, D. M. : Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. biol. Chem. 247, 1106-1113 (1972) Szentivanyi, A. : The beta adrenergic theory of the atopic abnormality in bronchial asthma. J. Allergy clin. Immunol. 42, 203 - 232 (1968)
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) Szentivanyi, A., Fishel, C. W., Talmage, D. W. : Adrenaline mediation of histamine and serotonin hyperglycemia in normal mice and the absence of adrenaline-induced hyperglycemia in pertussis-sensitized mice. J. infect. Dis. 113, 86-98 (1963)
Received February 11 / Accepted May 31, 1976
