1P
CONTROL OF TISSUE CYCLIC AMP CONTENT IN BOVINE TRACHEAL SUBMUCOSAL GLANDS
I. P. Hall & S. Widdop (introduced by P.C.. Rubin), Department of Therapeutics,.University Hospital of Nottingham, Nottingham NG7 2UH A mucus secretory response in airway submucosal glands can be elicited both by agents which in other tissues elevate intracellular calcium levels and by agents which elevate tissue cyclic AMP content implying the involvement of-two different second messenger pathways in the secretory response. We have previously demonstrated that a range of
secretagogues including carbachol and histamine are able to induce an inositol phosphate response in bovine tracheal submucosal glands (Hall 1992). In this study we have examined the potential for a range of agents to elevate cyclic AMP levels in this tissue.
Bovine tracheal submucosal gland slices were prepared and (3H]-cyclic AMP formation over 10 mins was quantified using the methods previously described(Hall et al 1989,Hall 1992).
Forskolin produced concentration related [3H]-cyclic AMP formation (nonmaximal response; response to luM 6.5 + 0.8 fold over basal, n=5). Histamine (EC50 7.6 + 1.2uM, n=3, maximum response 5.3 + 0.8 fold, n=6) and isoprenaline (EC50 20 + 5nM, n=3, maximum response 2.9 + 0.3 fold) both produced concentration related cyclic AMP formation in the presence of lmM 3-isobutyl-1-methylxanthine (IBMX). The response to histamine was inhibited by cimetidine (lOOuM; KA 1.3 + 0.2c1016M ), and the response to isoprenaline was inhibited by ICI 118551 (5OnM, KA 3 + 0.5x10 M ) indicating the involvement of H2 and beta 2 receptors in these responses (both n=3). Both the nonselective PDE inhibitor IBMX (apparent EC50 133 + 27uM, n=4, nonmaximal response) and the type IV selective PDE isoenzyme inhibitor rolipram (EC50 1.4 + 0.5uM, n=4) produced concentration related [3H]cyclic.AMP formation in this tissue. The response to 1mM IBMX was 3.5 + 0.2 fold, (n=14), and to 0.1mM rolipram 3.5 + 0.3 fold (n=7). Neither zaprinast TO.lmM) nor the type III PDE isoenzyme inhibitor SK&F 94836 elevated cyclic AMP l4vels. These results demonstrate that in bovine airway submucosal glands tissue cyclic AMP content can be elevated by stimulation of histamine H2 and beta 2 adrenergic receptors, activation of adenylyl cyclase, and inhibition of type IV PDE activity. We are grateful for financial support from the Cystic Fibrosis Research Trust.
Hall, I.P. (1992). Am. J. Physiol. (in press). Hall, I.P., Donaldson, J. & Hill, S.J. (1989).
2P
Br. J. Pharmacol.
97., 603-613
COMPARISON OF THE EFFECTS OF TWO CALCIUM MODULATORS IN TRACHEA ISOLATED FROM NORMAL AND ALBUMIN-SENSITIZED GUINEA-PIGS
S. De Jonckheere & DJ. McCaig, School of Pharmacy, The Robert Gordon Institute of Technology, Schoolhill Aberdeen. AB9 1FR. Abnormal Ca2+ handling in airway smooth muscle has been suggested as a cause of the airway hyperreactivity characteristic of bronchial asthma. (Black et al, 1989). We have compared the effects of two calcium modulators on vagally-mediated constriction in trachea from normal guinea-pigs and albumin-sensitized guinea-pigs, which serve as a model for bronchial asthma. The drugs used were verapamil, a voltage-dependent Ca2+-channel blocker and trifluoperazine (TFP), a calmodulin antagonist. Tracheae, with attendant right vagus and recurrent laryngeal nerves were removed from untreated (UT), sham-sensitized (SS, saline vehicle only) or albumin-sensitized (AS, sensitizing injections i.p. and s.c. plus twice weekly inhalation of albumin for 3 weeks). The vagus nerve was stimulated at frequencies 1-50Hz for 5s with pulses of supramaximal voltage and lms duration. Responses were assessed as changes in intraluminal pressure (ILP) in the Krebs-filled trachea with the cyclo-oxygenase inhibitor flurbiprofen, 106M, present. Table 1. Percentage reduction in vagal responses at a frequency of 1Hz.(Values are mean+s.e.mean, n=5-7. from UT *pnotechisII-v>notechisll'2>>notechisII-i. The facts that (i) this order does not correlate with our observed maximal enzymatic rates; notechis II-v>notexin>notechis 11'2>>notechis II-i, and (ii) that notechis 11'2 has a comparable enzymatic activity to non-myotoxic PLA2 homologues, support ..e postulated existence of regions upon PLA2 associated with the expression of neuro- and myotoxicity; these regions are presumably binding regions that attach the molecule to specific target tissues. Kini and Iwanaga (1986) have postulated that a cationic motif is associated with myotoxicity, but this motif is missing in the myotoxic compound notechis 11'2. Site directed and cassette mutagenesis experiments are currently underway using a cDNA clone encoding notechis 11'2 (Hodgson et al, unpublished). The aim is to insert into 11'2 sequences from notexin that may be associated with neurotoxicity (and hence lethality). Bouchier, C., Boyot, P., Tesson, F., Tremeau, Bouet, F., Hodgson, D., Boulain, J.-C. & Menez, A. (1991) Eur. J. Biochem. 202, 493-500. Harris, J.B. (1991) In Snake Toxins ed. Harvey, A.L. 91-129, Pergamon Press, New York. Kini, R.M. & Iwanaga, S. (1986' Toxicon 24, 895-905.
65P
EFFECT OF SR 58611A, A NOVEL ATYPICAL f3-ADRENOCEPTOR (p3) AGONIST, ON BROWN ADIPOSE TISSUE LIPOGENESIS
M. Al-Qatari and P. V. Taberner, Department of Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 lTD.
SR 58611A is a putative 83-adrenoceptor agonist phenylethanolaminotetraline (PEAT) developed for treating conditions of abnormally enchanced gastrointestinal motility (Bianchetti & Manara, 1990). CBA/Ca obese-diabetic mice exhibit reduced lipogenesis compared to normal littermates (Al-Qatari et. al., 1991). Brown adipose tissue (BAT) possesses atypical B adrenoceptors (B3) which stimulate energy expenditure by increasing fatty acid synthesis (lipogenesis) and thermogenesis. The aim of this study was to investigate the effect of SR 58611A on lipogenesis in adipose tissue of lean and obese-diabetic mice. In vivo lipogenesis rates were estimated in fed control (lean) and obese-diabetic male CBA/Ca mice by measuring the incorporation of 3H into fatty acids extracted from adipose tissue following i.p. injection of 3H20, using the methods Propranolol (10mg/kg) was given as i.p. injection 90 minutes prior to 3H20 described by Mercer & Trayhurn (1983). injection. SR 5861 1A (1 mg/kg i. p.) was administered acutely 1 hour and insulin (1 IU/kg i.p.) was injected 15 minutes prior to 3H20.
SR 58611A (1mg/kg) lowered BAT lipogenic rate slightly from control values ofll3.81±8.99(11) to 87.8±14.1(16) (means± S.E.M.(n)Itg atoms H incorporated/hr/g fat free tissue weight) in lean mice. However, in obese mice, lipogenic rates were increased significantly (p0.05). These pA2s for alprenolol differ from those previously reported for blockade of P- and P2-adrenoceptors in other tissues (8.1 and 8.0 respectively, Hoefle et al., 1975). These data further characterize the atypical p-adrenoceptors in GPGF, and show that clenbuterol, fenoterol and salbutamol possess only relatively weak agonist activity at these receptors.
Blue, D.R., Bond, R.A., Adham, N., Delmondo, R., Michel, A.D., Eglin, RM., Whiting, R.L. & Clarke, D.E. (1990). J. Pharm. Exp. Ther., 252 1034-1042. Coleman, RA., Denyer, L.H. & Sheldrick, K.E. (1987). Br. J. Pharmac., 90, 40P. Hoefle, Ml., Hastings, S.G., Meyer, R.F., Corey, RM., Holmes, A. & Stratton, C.D. (1975), J. Med. Chem., 18, 148-152.
DRUG EFFECTS ON VASCULAR FLOW IN THE ISOLATED PERFUSED BOVINE EYE USING RADIO-LABELLED MICROSPHERES
67P
C. Millar, R.D. Carr1, B.G. Humphries1 & W.S. Wilson, Department of Pharmacology, University of Glasgow, Glasgow, G12 8QQ, and 1Department of Pharmacology, Fisons plc, Loughborough, Leics, LE11 ORH We have previously reported (Millar & Wilson, 1991) the lowering of intraocular pressure (IOP) by sodium nitroprusside (SNP) and now show how its vasodilator activity extends to all the main ocular vascular via a long posterior ciliary artery and Within 50 min of slaughter, bovine eyes were beds. IOP was measured by cannulation of the perfused with an oxygenated [rebs solution at 2.25 ml .min- . At least 60 min was allowed for IOP to Tpilibrate; only eyes anterior chamber and a water manometer. Ce-labelled with a constant IOP in the range 6.5-12.5 m Hg ware used. Approximately 4,500 microspheres of 15 pm diameter were introduced into the arterial perfusate without disturbing the flow After 5 min the eye was dissected and each region weighed and counted for gamma radioactivity. rate.
cannulated
iris Timolol (30 nmol) significantly reduced IOP (Table 1.) but produced no change in perfusate flow in the or ciliary body, while a 300 nmol dose revealed an overall vasoconstrictor effect, though this was only significant in the iris. SNP significantly lowered IOP; while little vasod~latation was observed in the
absence of noradrenaline (NA; data not shown), arterial perfusion of NA (10-7 H) produced vascular tone (Table 1), against which significant vasodilatation occurred in all regions examined.
I
t12.
Dose
n
30 300
4,
Timolol
300
Control+ SNPS
104±46
4
2341 96 269± 541 37± 17'
183±19 89±37
73± 6 24± 6 52±10
5 5
30± 211a 240±103b
18±12a 117±7b
142± 7b
4
Control
Tisolol
Vascular flow (p1.Sin1 .1 tiasue) ciliary body choroid iris
(nmol)
& n
9 8 11
40±20
7
I01'
(mm Hg)
+0.1±0.41,
-2.2+0.3 -1.9±0.2" -2.0±0.3
Means ± s.e.mean. + Vascular flow measured in eyes perfused with [rebs containing NA (10-5 M). *P
CONTROL OF TISSUE CYCLIC AMP CONTENT IN BOVINE TRACHEAL SUBMUCOSAL GLANDS
I. P. Hall & S. Widdop (introduced by P.C.. Rubin), Department of Therapeutics,.University Hospital of Nottingham, Nottingham NG7 2UH A mucus secretory response in airway submucosal glands can be elicited both by agents which in other tissues elevate intracellular calcium levels and by agents which elevate tissue cyclic AMP content implying the involvement of-two different second messenger pathways in the secretory response. We have previously demonstrated that a range of
secretagogues including carbachol and histamine are able to induce an inositol phosphate response in bovine tracheal submucosal glands (Hall 1992). In this study we have examined the potential for a range of agents to elevate cyclic AMP levels in this tissue.
Bovine tracheal submucosal gland slices were prepared and (3H]-cyclic AMP formation over 10 mins was quantified using the methods previously described(Hall et al 1989,Hall 1992).
Forskolin produced concentration related [3H]-cyclic AMP formation (nonmaximal response; response to luM 6.5 + 0.8 fold over basal, n=5). Histamine (EC50 7.6 + 1.2uM, n=3, maximum response 5.3 + 0.8 fold, n=6) and isoprenaline (EC50 20 + 5nM, n=3, maximum response 2.9 + 0.3 fold) both produced concentration related cyclic AMP formation in the presence of lmM 3-isobutyl-1-methylxanthine (IBMX). The response to histamine was inhibited by cimetidine (lOOuM; KA 1.3 + 0.2c1016M ), and the response to isoprenaline was inhibited by ICI 118551 (5OnM, KA 3 + 0.5x10 M ) indicating the involvement of H2 and beta 2 receptors in these responses (both n=3). Both the nonselective PDE inhibitor IBMX (apparent EC50 133 + 27uM, n=4, nonmaximal response) and the type IV selective PDE isoenzyme inhibitor rolipram (EC50 1.4 + 0.5uM, n=4) produced concentration related [3H]cyclic.AMP formation in this tissue. The response to 1mM IBMX was 3.5 + 0.2 fold, (n=14), and to 0.1mM rolipram 3.5 + 0.3 fold (n=7). Neither zaprinast TO.lmM) nor the type III PDE isoenzyme inhibitor SK&F 94836 elevated cyclic AMP l4vels. These results demonstrate that in bovine airway submucosal glands tissue cyclic AMP content can be elevated by stimulation of histamine H2 and beta 2 adrenergic receptors, activation of adenylyl cyclase, and inhibition of type IV PDE activity. We are grateful for financial support from the Cystic Fibrosis Research Trust.
Hall, I.P. (1992). Am. J. Physiol. (in press). Hall, I.P., Donaldson, J. & Hill, S.J. (1989).
2P
Br. J. Pharmacol.
97., 603-613
COMPARISON OF THE EFFECTS OF TWO CALCIUM MODULATORS IN TRACHEA ISOLATED FROM NORMAL AND ALBUMIN-SENSITIZED GUINEA-PIGS
S. De Jonckheere & DJ. McCaig, School of Pharmacy, The Robert Gordon Institute of Technology, Schoolhill Aberdeen. AB9 1FR. Abnormal Ca2+ handling in airway smooth muscle has been suggested as a cause of the airway hyperreactivity characteristic of bronchial asthma. (Black et al, 1989). We have compared the effects of two calcium modulators on vagally-mediated constriction in trachea from normal guinea-pigs and albumin-sensitized guinea-pigs, which serve as a model for bronchial asthma. The drugs used were verapamil, a voltage-dependent Ca2+-channel blocker and trifluoperazine (TFP), a calmodulin antagonist. Tracheae, with attendant right vagus and recurrent laryngeal nerves were removed from untreated (UT), sham-sensitized (SS, saline vehicle only) or albumin-sensitized (AS, sensitizing injections i.p. and s.c. plus twice weekly inhalation of albumin for 3 weeks). The vagus nerve was stimulated at frequencies 1-50Hz for 5s with pulses of supramaximal voltage and lms duration. Responses were assessed as changes in intraluminal pressure (ILP) in the Krebs-filled trachea with the cyclo-oxygenase inhibitor flurbiprofen, 106M, present. Table 1. Percentage reduction in vagal responses at a frequency of 1Hz.(Values are mean+s.e.mean, n=5-7. from UT *pnotechisII-v>notechisll'2>>notechisII-i. The facts that (i) this order does not correlate with our observed maximal enzymatic rates; notechis II-v>notexin>notechis 11'2>>notechis II-i, and (ii) that notechis 11'2 has a comparable enzymatic activity to non-myotoxic PLA2 homologues, support ..e postulated existence of regions upon PLA2 associated with the expression of neuro- and myotoxicity; these regions are presumably binding regions that attach the molecule to specific target tissues. Kini and Iwanaga (1986) have postulated that a cationic motif is associated with myotoxicity, but this motif is missing in the myotoxic compound notechis 11'2. Site directed and cassette mutagenesis experiments are currently underway using a cDNA clone encoding notechis 11'2 (Hodgson et al, unpublished). The aim is to insert into 11'2 sequences from notexin that may be associated with neurotoxicity (and hence lethality). Bouchier, C., Boyot, P., Tesson, F., Tremeau, Bouet, F., Hodgson, D., Boulain, J.-C. & Menez, A. (1991) Eur. J. Biochem. 202, 493-500. Harris, J.B. (1991) In Snake Toxins ed. Harvey, A.L. 91-129, Pergamon Press, New York. Kini, R.M. & Iwanaga, S. (1986' Toxicon 24, 895-905.
65P
EFFECT OF SR 58611A, A NOVEL ATYPICAL f3-ADRENOCEPTOR (p3) AGONIST, ON BROWN ADIPOSE TISSUE LIPOGENESIS
M. Al-Qatari and P. V. Taberner, Department of Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 lTD.
SR 58611A is a putative 83-adrenoceptor agonist phenylethanolaminotetraline (PEAT) developed for treating conditions of abnormally enchanced gastrointestinal motility (Bianchetti & Manara, 1990). CBA/Ca obese-diabetic mice exhibit reduced lipogenesis compared to normal littermates (Al-Qatari et. al., 1991). Brown adipose tissue (BAT) possesses atypical B adrenoceptors (B3) which stimulate energy expenditure by increasing fatty acid synthesis (lipogenesis) and thermogenesis. The aim of this study was to investigate the effect of SR 58611A on lipogenesis in adipose tissue of lean and obese-diabetic mice. In vivo lipogenesis rates were estimated in fed control (lean) and obese-diabetic male CBA/Ca mice by measuring the incorporation of 3H into fatty acids extracted from adipose tissue following i.p. injection of 3H20, using the methods Propranolol (10mg/kg) was given as i.p. injection 90 minutes prior to 3H20 described by Mercer & Trayhurn (1983). injection. SR 5861 1A (1 mg/kg i. p.) was administered acutely 1 hour and insulin (1 IU/kg i.p.) was injected 15 minutes prior to 3H20.
SR 58611A (1mg/kg) lowered BAT lipogenic rate slightly from control values ofll3.81±8.99(11) to 87.8±14.1(16) (means± S.E.M.(n)Itg atoms H incorporated/hr/g fat free tissue weight) in lean mice. However, in obese mice, lipogenic rates were increased significantly (p0.05). These pA2s for alprenolol differ from those previously reported for blockade of P- and P2-adrenoceptors in other tissues (8.1 and 8.0 respectively, Hoefle et al., 1975). These data further characterize the atypical p-adrenoceptors in GPGF, and show that clenbuterol, fenoterol and salbutamol possess only relatively weak agonist activity at these receptors.
Blue, D.R., Bond, R.A., Adham, N., Delmondo, R., Michel, A.D., Eglin, RM., Whiting, R.L. & Clarke, D.E. (1990). J. Pharm. Exp. Ther., 252 1034-1042. Coleman, RA., Denyer, L.H. & Sheldrick, K.E. (1987). Br. J. Pharmac., 90, 40P. Hoefle, Ml., Hastings, S.G., Meyer, R.F., Corey, RM., Holmes, A. & Stratton, C.D. (1975), J. Med. Chem., 18, 148-152.
DRUG EFFECTS ON VASCULAR FLOW IN THE ISOLATED PERFUSED BOVINE EYE USING RADIO-LABELLED MICROSPHERES
67P
C. Millar, R.D. Carr1, B.G. Humphries1 & W.S. Wilson, Department of Pharmacology, University of Glasgow, Glasgow, G12 8QQ, and 1Department of Pharmacology, Fisons plc, Loughborough, Leics, LE11 ORH We have previously reported (Millar & Wilson, 1991) the lowering of intraocular pressure (IOP) by sodium nitroprusside (SNP) and now show how its vasodilator activity extends to all the main ocular vascular via a long posterior ciliary artery and Within 50 min of slaughter, bovine eyes were beds. IOP was measured by cannulation of the perfused with an oxygenated [rebs solution at 2.25 ml .min- . At least 60 min was allowed for IOP to Tpilibrate; only eyes anterior chamber and a water manometer. Ce-labelled with a constant IOP in the range 6.5-12.5 m Hg ware used. Approximately 4,500 microspheres of 15 pm diameter were introduced into the arterial perfusate without disturbing the flow After 5 min the eye was dissected and each region weighed and counted for gamma radioactivity. rate.
cannulated
iris Timolol (30 nmol) significantly reduced IOP (Table 1.) but produced no change in perfusate flow in the or ciliary body, while a 300 nmol dose revealed an overall vasoconstrictor effect, though this was only significant in the iris. SNP significantly lowered IOP; while little vasod~latation was observed in the
absence of noradrenaline (NA; data not shown), arterial perfusion of NA (10-7 H) produced vascular tone (Table 1), against which significant vasodilatation occurred in all regions examined.
I
t12.
Dose
n
30 300
4,
Timolol
300
Control+ SNPS
104±46
4
2341 96 269± 541 37± 17'
183±19 89±37
73± 6 24± 6 52±10
5 5
30± 211a 240±103b
18±12a 117±7b
142± 7b
4
Control
Tisolol
Vascular flow (p1.Sin1 .1 tiasue) ciliary body choroid iris
(nmol)
& n
9 8 11
40±20
7
I01'
(mm Hg)
+0.1±0.41,
-2.2+0.3 -1.9±0.2" -2.0±0.3
Means ± s.e.mean. + Vascular flow measured in eyes perfused with [rebs containing NA (10-5 M). *P
