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Beta-Adrenergic Modulation of Airway Epithelial Glycoprotein
Beta-Adrenergic Modulation of Airway Epithelial Glycoprotein* Rosemary ]ones, Ph.D.; and Lynne Reid, M.D.
T
he structure of extracellula~!glycoprotein molecules in airway mucus depends upon intracellular patterns of synthesis. In airway secretory cells, granules are all one type of glycoprotein, or granules may be mixed. The glycoprotein may be neutral or acid, and when acid, sulfated or sialylated ( sialidase sensitive or resistant) . Little is known of the way intracellular glycoprotein and secretory cell concentration are modulated in the normal airway, or in disease associated with mucus hypersecretion. In the studies reported here, our aim is to elucidate mechanisms of control of the epithelial cell population and the glycoprotein type at various airway lavels. We describe in rats 1) the speed and pattern of secretory cell hyperplasia, 2) the modulation of intracellular glycoprotein, after administration of the ,a-adrenergic agonist isoproterenol, and 3) the regression of these changes after withdrawal of the drug. METHODS
Adult male Sprague-Dawley rats were injected subcutaneously with dl-isoproterenol hydrochloride ( 10 mg/ 100 g body weight for 1-6 days) ; control animals were injected with normal saline solution. 1 Twenty-four hours, or l-12 weeks, after the last injection of drug or saline solution, the lungs were excised and distended with formol saline. Using quantitative histochemical techniques to demonstrate intracellular glycoprotein ( s) , secretory cell concentration and intracellular glycoprotein( s) were examined in airway epithelium from larynx to lung periphery." The regions included proximal (level I) and distal trachea (level 3a), left main bronchus (level 3b), proximal and distal regions of the main intrapulmonary airway (levels 4 and 5) and two of its lateral branches (levels 6 and 7). 3 The mid-tracheal region (level 2) is not described here since isoproterenol does not significantly increase the secretory cell concentration , although it causes a significant shift in intracellular glycoprotein.1 Regression of changes was followed after the maximum time of drug administration, ie 6 days.
nCON
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Extrapulmonary
VI
Intrapulmonary
FIGURE 1. The effect of a single injection of isoproterenol (IPR) or normal saline (CON) on secretory cell concentration in 7 regions of rat airway from larynx to lung periphery (mean ± SE). tion of isoproterenol significantly increased the secretory cell concentration in all regions except the most proximal (level 1 ) , where it remained similar to the control value (Fig 1). In response to the first injectrl>n, the relative increase in concentration was greatest at the periphery ( 6 to 8-fold, levels 5 and 7 respectively). After a second injection, the concentration in each region was significantly above the control and it remained significantly increased after further injections. Regional patterns of increase were maintained. During the series of drug injections a significant shift in distribution of secretory cell types from control patterns {Px 2 <0.05) was seen in all except one peripheral region {level 5) . In all regions, a single injection of drug increased cells containing acid glycoprotein, either as the only granule type within a cell or mixed with granules of neutral glycoprotein. In the most proximal region (level 1 ) further drug administration increased cells synthesizing neutral glycoprotein; in other regions increase was in cells synthesizing acid glycoprotein that was either sulfated or sialidase-resistant sialylated.
=
E :J
l..eYel III b
240-
0 CON
Qj
.
RESULTS
Both in response to isoproterenol and in recovery there is regional airway variation in secretory cell increase and in modification of intracellular glycoprotein.
Speed and Pattern of Development of Airway Changes
VII
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200-
E
160-
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120-
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0
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In each airway region of control animals injected with normal saline solution, the secretory cell concentration varied somewhat over the 6-day period of administration, but in no region was it significantly different at the end of the period from the start. A single injec-
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0Department of Pathology~ The Children's Hospital Medical Center, Harvard Meclical School, Boston. Reprint requests: Dr. Jones, Department of Pathology, 300 Longwood Avenue, Boston 02115
FIGURE 2. Secretory cell concentration in rat airway (level 3b-left main bronchus) 24 hours and 1-12 weeks after stopping administration of isoproterenol ( IPR) or normal saline (CON) (mean± SE) .
CHEST, 81: 5, MAY, 1982 SUPPLEMENT
Ill
0 24hrs
1
2
3
4
6
8
12
Recovery {weeks )
LUNG FLUIDS AND SECRETIONS 25S
Speed and Pattern of Recovery of Airway Changes Regression was followed in 5 airway regions (levels 1-5). During recovery from isoproterenol administration the secretory cell concentration fell faster in some regions than in others. In no region was the fall steady. One week after stopping drug injections the concentration was normal proximally (level 1 ) and here it remained close to control values. In other regions, during regression, concentration fluctuated. The pattern in one central region (level 3b) is illustrated (Fig 2) . Mter 1 week of recovery, the concentration in this region was still 94% of its value 24 hours after the last injection of isoproterenol. Mter 2 weeks of recovery it fell to 53% and it remained at this level until, after 8 weeks, it fell again, to 27%. Mter 12 weeks it was 22% of its original value and close to the control value at the start of recovery. Since, in this region, the control value also fell somewhat during recovery (see Fig 2), and was least at the end, the final secretory cell concentration in the airways of animals recovering from drug administration remained high. The pattern in other airway regions was similar (levels 3a, 4 and 5), although some regional differences were apparent. As judged by intracellular granules of glycoprotein, the distribution of secretory cells in each airway region of animals recovering from isoproterenol treatment fluctuated during the 12-week recovery period. In the proximal region (level 1) the distribution was normal by 1 week, and in one distal extrapulmonary region (level 3a) , normal by 2 weeks. In other regions, the number of cells synthesizing acid glycoprotein fell to equal the control value by 8 weeks, but cells synthesizing neutral glycoprotein increased during recovery. DISCUSSION
In response to isoproterenol stimulation, the regional pattern of secretory cells in normal rat airway, both concentration and glycoprotein type, is lost. Synthesis of glycoprotein in the new secretory cells appearing in response to drug probably results from activation of galactosyltransferases, fucosyltransferases, sialyltransferases and possibly sulfotransferases. Whether this change within the airway cell population is produced directly, or indirectly, by the drug or its metabolites, or through release of another mediator, is not known. Current evidence suggests that the P-adrenoceptor is located on the plasma membrane and indirectly coupled to adenylate cyclase. • Activation of this enzyme by the formation of a drug/ receptor complex catalyzes the receptor for adenosine triphosphate (ATP) on the inner surface of the plasma membrane, converting ATP to cyclic adenosine monophosphate (cAMP) .5 Several ions are important to the reaction, eg, magnesium (needed for adenylate cyclase to be effective) and calcium (influx inhibiting action of the enzyme). It is likely that isoproterenol exerts its effect through activation of adenylate cyclase, so increasing the level of cAMP. McMahon 6
26S 24TH ASPEN LUNG CONFERENCE
considers that cell differentiation is controlled by cyclic nucleotides, in the developing lung as well as in the mature lung, as do Cutler and Rhodin. 7 In the present study it is in the mature lung that differentiation or change in cell type is seen in response to isoproterenol treatment. In that the changes regress, evidently continued administration of drug is needed to maintain them. That they do not regress quickly in most regions suggests that switch-off in glycoprotein synthesis is not as readily achieved as switch-on, or perhaps that cells have become more susceptible to endogenous levels of mediators or other homeostatic mechanisms. In the normal airway, mechanisms for homeostasis must exist to maintain regional differences in epithelial cell concentration, epithelial cell type and frequency, as well as epithelial cell replacement. Since isoproterenol resembles epinephrine and norepinephrine it may mimic endogenous mediators, the potency of the drug determining its greater response. 5 •8 In addition to P-adrenoceptors it is likely that a-adrenergic and cholinergic receptors9 • 10 are present on airway epithelial cells and further studies are needed to demonstrate their presence and to elucidate their interaction. While the P-adrenergic system doubtless represents only part of the control of the airway surface epithelium, it represents an important part, for its possible role in normal homeostasis, its disturbance in temporary adaptation to an acute stimulus and its more chronic disturbance in disease. REFERENCES
2 3
4 5 6 7
8 9
10
Jones R, Reid L. p-agonists and secretory cell number and intracellular glycoprotein in airway epithelium: the effect of isoproterenol and salbutamol. Am J Pathol 1979; 95 :407-21 Jones R, Reid L. Secretory cells and their glycoproteins in health and disease. Br Med Bull 1978; 34 :9-16 Jones R, Reid L. Secretory cell hyperplasia and modification of intracellular glycoprotein in the rat airways induced by short periods of exposure to tobacco smoke and the effect of the anti-inflammatory agent phenylmethyloxadiazole. Lab Invest 1978; 39:41-49 Williams L T, Lefkowitz RL. Receptor binding studies in adrenergic pharmacology. New York : Raven Press, 1978 Brittain RT, Dean CM, Jack D. Sympathomimetic bronchodilator drugs. Pharm Therap 1976; B2 :423-62 McMahon D. Chemical messengers in development: a hypothesis. Science, 1974; 185 :1012-21 Cutler LS, Rhodin SB. Biochemical and cytochemical studies on adenylate cyclase activity in the developing rat submandibular gland: differentiation of the acinar secretory compartment. J Embryo! Exp Morph, 1976; 36:291-303 Hartley D, Jack D. Selectivity of action in ,6-adrenoceptor stimulants. J Appl Chern Biotech 1978; 28 :173-82 Sturgess J, Reid L . The effect of isoprenaline and pilocarpine on ( a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen. Br J Exp Pathol 1973; 54:388-403 Yoneda K. Pilocarpine stimulation of the bronchiolar Clara cell secretion. Lab Invest 1977; 37:447-52
CHEST, 81: 5, MAY, 1982 SUPPLEMENT
T
he structure of extracellula~!glycoprotein molecules in airway mucus depends upon intracellular patterns of synthesis. In airway secretory cells, granules are all one type of glycoprotein, or granules may be mixed. The glycoprotein may be neutral or acid, and when acid, sulfated or sialylated ( sialidase sensitive or resistant) . Little is known of the way intracellular glycoprotein and secretory cell concentration are modulated in the normal airway, or in disease associated with mucus hypersecretion. In the studies reported here, our aim is to elucidate mechanisms of control of the epithelial cell population and the glycoprotein type at various airway lavels. We describe in rats 1) the speed and pattern of secretory cell hyperplasia, 2) the modulation of intracellular glycoprotein, after administration of the ,a-adrenergic agonist isoproterenol, and 3) the regression of these changes after withdrawal of the drug. METHODS
Adult male Sprague-Dawley rats were injected subcutaneously with dl-isoproterenol hydrochloride ( 10 mg/ 100 g body weight for 1-6 days) ; control animals were injected with normal saline solution. 1 Twenty-four hours, or l-12 weeks, after the last injection of drug or saline solution, the lungs were excised and distended with formol saline. Using quantitative histochemical techniques to demonstrate intracellular glycoprotein ( s) , secretory cell concentration and intracellular glycoprotein( s) were examined in airway epithelium from larynx to lung periphery." The regions included proximal (level I) and distal trachea (level 3a), left main bronchus (level 3b), proximal and distal regions of the main intrapulmonary airway (levels 4 and 5) and two of its lateral branches (levels 6 and 7). 3 The mid-tracheal region (level 2) is not described here since isoproterenol does not significantly increase the secretory cell concentration , although it causes a significant shift in intracellular glycoprotein.1 Regression of changes was followed after the maximum time of drug administration, ie 6 days.
nCON
E 200-
:I
-
160-
E
120-
'ii
s=
~ IPR
0.
~ Gl
u
-
Gl
E
...0>-,.,c ...u
80-
Gl Gl
VI
40-
0
z
~
Level : I
rna
v
IV
lllb
Extrapulmonary
VI
Intrapulmonary
FIGURE 1. The effect of a single injection of isoproterenol (IPR) or normal saline (CON) on secretory cell concentration in 7 regions of rat airway from larynx to lung periphery (mean ± SE). tion of isoproterenol significantly increased the secretory cell concentration in all regions except the most proximal (level 1 ) , where it remained similar to the control value (Fig 1). In response to the first injectrl>n, the relative increase in concentration was greatest at the periphery ( 6 to 8-fold, levels 5 and 7 respectively). After a second injection, the concentration in each region was significantly above the control and it remained significantly increased after further injections. Regional patterns of increase were maintained. During the series of drug injections a significant shift in distribution of secretory cell types from control patterns {Px 2 <0.05) was seen in all except one peripheral region {level 5) . In all regions, a single injection of drug increased cells containing acid glycoprotein, either as the only granule type within a cell or mixed with granules of neutral glycoprotein. In the most proximal region (level 1 ) further drug administration increased cells synthesizing neutral glycoprotein; in other regions increase was in cells synthesizing acid glycoprotein that was either sulfated or sialidase-resistant sialylated.
=
E :J
l..eYel III b
240-
0 CON
Qj
.
RESULTS
Both in response to isoproterenol and in recovery there is regional airway variation in secretory cell increase and in modification of intracellular glycoprotein.
Speed and Pattern of Development of Airway Changes
VII
...
.!! u
..
>-
...u.
~ Q.
200-
E
160-
~
120-
..,E
0
80-
•
40-
~ IPR
In each airway region of control animals injected with normal saline solution, the secretory cell concentration varied somewhat over the 6-day period of administration, but in no region was it significantly different at the end of the period from the start. A single injec-
z
0Department of Pathology~ The Children's Hospital Medical Center, Harvard Meclical School, Boston. Reprint requests: Dr. Jones, Department of Pathology, 300 Longwood Avenue, Boston 02115
FIGURE 2. Secretory cell concentration in rat airway (level 3b-left main bronchus) 24 hours and 1-12 weeks after stopping administration of isoproterenol ( IPR) or normal saline (CON) (mean± SE) .
CHEST, 81: 5, MAY, 1982 SUPPLEMENT
Ill
0 24hrs
1
2
3
4
6
8
12
Recovery {weeks )
LUNG FLUIDS AND SECRETIONS 25S
Speed and Pattern of Recovery of Airway Changes Regression was followed in 5 airway regions (levels 1-5). During recovery from isoproterenol administration the secretory cell concentration fell faster in some regions than in others. In no region was the fall steady. One week after stopping drug injections the concentration was normal proximally (level 1 ) and here it remained close to control values. In other regions, during regression, concentration fluctuated. The pattern in one central region (level 3b) is illustrated (Fig 2) . Mter 1 week of recovery, the concentration in this region was still 94% of its value 24 hours after the last injection of isoproterenol. Mter 2 weeks of recovery it fell to 53% and it remained at this level until, after 8 weeks, it fell again, to 27%. Mter 12 weeks it was 22% of its original value and close to the control value at the start of recovery. Since, in this region, the control value also fell somewhat during recovery (see Fig 2), and was least at the end, the final secretory cell concentration in the airways of animals recovering from drug administration remained high. The pattern in other airway regions was similar (levels 3a, 4 and 5), although some regional differences were apparent. As judged by intracellular granules of glycoprotein, the distribution of secretory cells in each airway region of animals recovering from isoproterenol treatment fluctuated during the 12-week recovery period. In the proximal region (level 1) the distribution was normal by 1 week, and in one distal extrapulmonary region (level 3a) , normal by 2 weeks. In other regions, the number of cells synthesizing acid glycoprotein fell to equal the control value by 8 weeks, but cells synthesizing neutral glycoprotein increased during recovery. DISCUSSION
In response to isoproterenol stimulation, the regional pattern of secretory cells in normal rat airway, both concentration and glycoprotein type, is lost. Synthesis of glycoprotein in the new secretory cells appearing in response to drug probably results from activation of galactosyltransferases, fucosyltransferases, sialyltransferases and possibly sulfotransferases. Whether this change within the airway cell population is produced directly, or indirectly, by the drug or its metabolites, or through release of another mediator, is not known. Current evidence suggests that the P-adrenoceptor is located on the plasma membrane and indirectly coupled to adenylate cyclase. • Activation of this enzyme by the formation of a drug/ receptor complex catalyzes the receptor for adenosine triphosphate (ATP) on the inner surface of the plasma membrane, converting ATP to cyclic adenosine monophosphate (cAMP) .5 Several ions are important to the reaction, eg, magnesium (needed for adenylate cyclase to be effective) and calcium (influx inhibiting action of the enzyme). It is likely that isoproterenol exerts its effect through activation of adenylate cyclase, so increasing the level of cAMP. McMahon 6
26S 24TH ASPEN LUNG CONFERENCE
considers that cell differentiation is controlled by cyclic nucleotides, in the developing lung as well as in the mature lung, as do Cutler and Rhodin. 7 In the present study it is in the mature lung that differentiation or change in cell type is seen in response to isoproterenol treatment. In that the changes regress, evidently continued administration of drug is needed to maintain them. That they do not regress quickly in most regions suggests that switch-off in glycoprotein synthesis is not as readily achieved as switch-on, or perhaps that cells have become more susceptible to endogenous levels of mediators or other homeostatic mechanisms. In the normal airway, mechanisms for homeostasis must exist to maintain regional differences in epithelial cell concentration, epithelial cell type and frequency, as well as epithelial cell replacement. Since isoproterenol resembles epinephrine and norepinephrine it may mimic endogenous mediators, the potency of the drug determining its greater response. 5 •8 In addition to P-adrenoceptors it is likely that a-adrenergic and cholinergic receptors9 • 10 are present on airway epithelial cells and further studies are needed to demonstrate their presence and to elucidate their interaction. While the P-adrenergic system doubtless represents only part of the control of the airway surface epithelium, it represents an important part, for its possible role in normal homeostasis, its disturbance in temporary adaptation to an acute stimulus and its more chronic disturbance in disease. REFERENCES
2 3
4 5 6 7
8 9
10
Jones R, Reid L. p-agonists and secretory cell number and intracellular glycoprotein in airway epithelium: the effect of isoproterenol and salbutamol. Am J Pathol 1979; 95 :407-21 Jones R, Reid L. Secretory cells and their glycoproteins in health and disease. Br Med Bull 1978; 34 :9-16 Jones R, Reid L. Secretory cell hyperplasia and modification of intracellular glycoprotein in the rat airways induced by short periods of exposure to tobacco smoke and the effect of the anti-inflammatory agent phenylmethyloxadiazole. Lab Invest 1978; 39:41-49 Williams L T, Lefkowitz RL. Receptor binding studies in adrenergic pharmacology. New York : Raven Press, 1978 Brittain RT, Dean CM, Jack D. Sympathomimetic bronchodilator drugs. Pharm Therap 1976; B2 :423-62 McMahon D. Chemical messengers in development: a hypothesis. Science, 1974; 185 :1012-21 Cutler LS, Rhodin SB. Biochemical and cytochemical studies on adenylate cyclase activity in the developing rat submandibular gland: differentiation of the acinar secretory compartment. J Embryo! Exp Morph, 1976; 36:291-303 Hartley D, Jack D. Selectivity of action in ,6-adrenoceptor stimulants. J Appl Chern Biotech 1978; 28 :173-82 Sturgess J, Reid L . The effect of isoprenaline and pilocarpine on ( a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen. Br J Exp Pathol 1973; 54:388-403 Yoneda K. Pilocarpine stimulation of the bronchiolar Clara cell secretion. Lab Invest 1977; 37:447-52
CHEST, 81: 5, MAY, 1982 SUPPLEMENT