Br. J. Pharmacol. (1992), 105, 603-608

.v,'-. Macmillan Press Ltd, 1992

Studies on the mechanism of 5-HT1 receptor-induced smooth muscle contraction in dog saphenous vein 1Michael J. Sumner, Wasyl Feniuk, Julie D. McCormick & Patrick P.A. Humphrey Pharmacology Division, Glaxo Group Research Ltd., Ware, Hertfordshire SG12 ODP 1 We have investigated the mechanism of smooth muscle contraction evoked by activation of 5-HT1-like receptors in dog isolated saphenous vein. 2 In the presence of the 5-HT2 receptor antagonist, ritanserin (0.1pM), concentration-effect

curves

(lOnM-300pM) for 5-hydroxytryptamine (5-HT)-induced smooth muscle contraction were biphasic. This could be attributed to a direct action on 5-HT1-like receptors at low concentrations of 5-HT (10nm1OpM) and an indirect (through the release of noradrenaline from sympathetic neurones) activation of postjunctional a-adrenoceptors at higher 5-HT concentrations. In contrast, concentration-effect curves (100nM-tOOpM) for sumatriptan-induced contractions were not biphasic, and were due solely to activation of 5-HT1-like receptors. 3 Smooth muscle contractions evoked either by low concentrations of 5-HT or by sumatriptan were abolished by removal of extracellular calcium and were markedly inhibited, but not abolished, by the calcium channel blocker, verapamil (1-30pM). In contrast, contractions evoked by high concentrations of 5-HT were markedly less sensitive to removal of extracellular calcium or to verapamil. 4 5-HT and sumatriptan also inhibited (to a maximum of about 50%) prostaglandin E2 (PGE2 ,5 pM)stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation. This effect was mimicked by the a2-adrenoceptor agonist, azepexole (B-HT933) but not by the a1-adrenoceptor agonist, methoxamine. 5 In contrast to mediation of smooth muscle contraction, the 5-HT1-like receptor-mediated inhibition of PGE2-stimulated cyclic AMP formation evoked by 5-HT or sumatriptan was not attenuated by removal of extracellular calcium or by verapamil (1 PM). 6 A directly-acting inhibitor of adenylyl cyclase, 2',3'-dideoxyadenosine (1 mM) inhibited PGE2-stimulated cyclic AMP formation but did not produce smooth muscle contraction. 7 These results suggest that contractile responses of dog isolated saphenous vein arising through activation of 5-HT1-like receptors are associated with both an influx of extracellular calcium ions (to a large extent via voltage-dependent channels) and an inhibition of adenylyl cyclase. However, although these two responses are coupled to the same receptor, they appear to be independent. Keywords: Sumatriptan; 5-HT1-like receptor; dog saphenous vein; adenylyl cyclase; calcium; cyclic AMP

Introduction Sumatriptan is a novel and selective 5-HT1-like receptor agonist (Humphrey et al., 1988) which is clinically effective in the treatment of migraine (Patten, 1991). Sumatriptan produces both smooth muscle contraction (Humphrey et al., 1988) and inhibition of prostaglandin E2 (PGE2)stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation (Sumner & Humphrey, 1990) in dog isolated saphenous vein. We have attempted to explore further the mechanism whereby sumatriptan evokes smooth muscle contraction in the dog saphenous vein preparation. In this regard, the roles of intracellular and extracellular calcium in the contractile responses elicited by al- and a2-adrenoceptor agonists have been investigated previously (Mathews et al., 1984; reviewed in Biilbring & Tomita, 1987 and Docherty, 1989). These studies have demonstrated that smooth muscle contraction evoked by a2-adrenoceptor activation is more sensitive to calcium channel bockers, both in vitro (Mathews et al., 1984) and in vivo (Van Meel et al., 1983), than is that evoked through activation of al-adrenoceptors. Although the receptor-coupling mechanisms underlying these responses in dog saphenous vein are not known, in other tissues, al- and x2-adrenoceptors appear to be coupled to activation of phospholipase C or to inhibition of adenylyl cyclase respectively (Fain & Garcia-Sainz, 1980). In keeping with this, a2-adrenoceptor-mediated pressor responses in vivo can be attenuated by pretreatment with pertussis toxin (Boyer et al., would suggest that 1983). These observations Author for correspondence.

x2-adrenoceptor-mediated smooth muscle contraction may result from the influx of extracellular calcium (largely through voltage-dependent channels) and the inhibition of adenylyl cyclase. We therefore sought to explore whether the same may be true for 5-HT1-like receptor-mediated smooth muscle contraction in dog isolated saphenous vein. In addition, sumatriptan has also been shown to act as an agonist at brain 5-HT1D receptors (Schoeffter & Hoyer, 1989). These receptors are negatively coupled to adenylyl cyclase (Waeber et al., 1988; Schoeffter & Hoyer, 1989) and as such are somewhat similar to dog saphenous vein 5-HT1-like receptors (Summer & Humphrey, 1990). Accordingly, we have attempted to characterize in more detail the receptor mediating inhibition of adenylyl cyclase in dog saphenous vein with a view to comparing it with brain 5-HTlD receptors. Methods Lengths (3-5 cm) of lateral saphenous vein were removed from Beagle dogs of either sex (7-10 kg body weight) which had been anaesthetized with barbitone (300mgkg-1, i.p.) following induction with thiopentone (25mgkg- i.v.) and pentobarbitone (60mg, i.v.). These blood vessels were then used for measurements of smooth muscle contraction or cyclic AMP formation.

Measurement of smooth muscle contraction was performed essentially as described by Apperley et al. (1980). Briefly, four spirally-cut strips (2 mm wide, 2-3 cm

This

long) were mounted individually in 20 ml organ baths for isometric recording of tension changes and were maintained at

604

M.J. SUMNER et al.

* 370C in a gassed (95% 02, 5% CG2), modified Krebs solution (composition, mM: NaCl 118, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 0.6, D-glucose 11.0, containing CaCl2 1.3 mm and ritanserin 0.1 pM (5-HT2 receptor antagonist)). Tissues were subjected to an initial resting tension of 0.5 g and were allowed to equilibrate for 60 min with changes of Krebs solution every 15 min. During this time, preparations were contracted once with a single concentration (30 mM) of KCI. Subsequently, a cumulative concentration-effect curve to either 5-hydroxytryptamine (5-HT) or sumatriptan was established for all preparations. In all cases, the preparations developed a sustained contraction and responses were measured at the plateau tension. In experiments exploring the calcium sensitivity of contractile responses to 5-HT or sumatriptan, the Krebs solution was then replaced with a similar solution containing either 2.6 mM, 1.3 mm, 0.65 mm, 0.33 mm or nominally 0 mm CaCl2 (not containing EGTA), and a cumulative concentration-effect curve to 5-HT or sumatriptan was repeated 30min later. In this way, cumulative concentration-effect curves for each agonist were generated in the presence of varying concentrations of calcium. Responses were then calculated as a percentage of the maximal contraction evoked during the determination of the original concentration-effect curve to 5-HT or sumatriptan (which was obtained in Krebs solution containing 1.3 mm CaCI2, a concentration which reflects plasma free calcium

concentrations). To examine the effects of verapamil, following an initial concentration-effect curve to 5-HT or sumatriptan, tissues were exposed for 30 min to vehicle (control) or verapamil (1, 3, 10 or 30pM) before an agonist concentration-effect curve was repeated.

Measurement of cyclic AMPformation Rings of dog isolated saphenous vein (2-3 mm in length) were incubated for 30min at 37°C in gassed (95% 02, 5% CG2), Krebs solution (1.3 mm CaCl2) containing the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 pM) and antagonists where appropriate. Rings were then co-incubated for 2 min at 37°C with 5 gM prostaglandin E2 (PGE2) and either vehicle (control) or test agonist, before being rapidly frozen in liquid nitrogen to terminate cyclic AMP formation. Rings were subsequently extracted for the measurement of protein content and cyclic AMP as previously described (Sumner et al., 1989). The cyclic AMP assay employed a commercially available kit (Amersham International, England) and was based upon the competition between [3H]-cyclic AMP and unlabelled cyclic AMP for a finite number of sites on a binding protein. Separation of bound and unbound cyclic AMP was achieved by charcoal sedimentation. The sensitivity of this assay (nonacetylated protocol) was 1-2 pmol per tube. Statistics Results are presented as arithmetic means with standard error of the mean from n observations, which is also the number of veins used. Student's unpaired t test was used to assess the significance of differences between mean values, significance being defined by a P value less than 0.05.

Drugs Drugs were obtained from the following sources: verapamil hydrochloride (Sigma), 2',3'-dideoxyadenosine (Sigma), 3isobutyl-1-methylxanthine (Sigma), forskolin (Sigma), spiperone ketanserin and ritanserin (Janssen), methiothepin maleate (Hoffman La-Roche), metergoline (Farmitalia), methysergide hydrogen maleate and mesulergine (Sandoz), azepexole (B-HT933; Boehringer-Ingelheim) and prostaglandin E2 (Upjohn). The following compounds were synthesized by Glaxo Group Research: sumatriptan, 5-carboxamidotryptamine, cyanopindolol and ondansetron.

Drug stocks were prepared and diluted in distilled water or Krebs buffer, with the exception of cyanopindolol, ketanserin, spiperone and ondansetron which were dissolved in IM acetic acid and diluted in Krebs buffer.

Results Smooth muscle contraction Concentration-effect curves for 5-HT (10nM-300pM)-induced contraction of strips of dog isolated saphenous vein were biphasic, the second phase being evident at 5-HT concentration greater than 10 uM (Figure 1). Decreasing the extracellular calcium concentration in the Krebs solution from 2.6 to Omm reduced the maximal contractile response to low concentrations of 5-HT in a concentration-dependent manner (Figure 1), with little or no response being evident in the absence of extracellular calcium. In contrast, contractions evoked by high concentrations of 5-HT were markedly less sensitive to decreasing the extracellular calcium concentration, with contractile responses still being evident even in the absence of calcium (Figure 1). In contrast to those obtained with 5-HT, concentrationeffect curves for sumatriptan (100nM-lOO1M)-induced smooth muscle contraction were not biphasic (Figure 2). The magnitude of the contraction induced by sumatriptan, like that evoked by low concentrations of 5-HT, was markedly reduced by decreasing the extracellular calcium concentration and was abolished in the absence of calcium (Figure 2). In the presence of 1.3mM calcium, verapamil (1-30pM) inhibited smooth muscle contraction evoked either by low concentrations of 5-HT (Figure 3) or by sumatriptan (Figure 4). However, this inhibition was not complete, with a substantial proportion of the response to 5-HT (low concentrations) or to sumatriptan being resistant to verapamil. Contractions evoked by high concentrations of 5-HT were less susceptible to blockade by verapamil, although some inhibition was apparent in the presence of 30 pM verapamil (Figure 3).

Inhibition of prostaglandin E2-stimulated cyclic AMP

formation Sumatriptan, 5-HT and 5-carboxamidotryptamine (5-CT) all inhibited (at 10 pM) PGE2-stimulated cyclic AMP formation 280 C

0 240 C

200-

0

E 160-

EX

120-

E C

80-

°

40 0

10-8

1O07

10-6

10-4

1i05

Concentration (mol

1O-3

1-1)

Figure 1 Effect of altering the extracellular calcium concentration on the contraction of dog isolated saphenous vein evoked by 5hydroxytryptamine (5-HT). An initial cumulative concentration-effect curve to 5-HT was established in the presence of 1.3 mM CaCl2. The Krebs solution was then replaced with one containing either 2.6mM (V) calcium and, (0), 1.3mM (M), 0.65mM (A), 0.33mM (Ol) or 0mM 30 min later, a cumulative concentration-effect curve to 5-HT was repeated. Results are mean of 4 determinations, and are expressed relative to the initial maximal response to 5-HT obtained in Krebs containing 1.3 mm CaCl2; vertical bars show s.e.mean. The 5-HT2 receptor antagonist, ritanserin (0.1 gM) was present throughout.

5-HT1 RECEPTOR-INDUCED SMOOTH MUSCLE CONTRACTION

.° 120

2 200 180-

co

: 100 _

C1600 E 140

0 0

E 120

1

-

100 80

E

E

80

M

60

E

C

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40 40

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0 Co

0 10-4 10-3 10-6 i05 Concentration (mol 1-1) Figure 2 Effects of altering extracellular calcium concentrations on the contraction of dog isolated saphenous vein by sumatriptan. An initial cumulative concentration-effect curve to sumatriptan was established on all preparations in the presence of 1.3 mM CaC12. The Krebs solution was then replaced with one containing 2.6mM (0), 1.3mM (S), 0.65mM (A) or OmM (V) calcium and, 30min later, a cumulative concentration-effect curve to sumatriptan was repeated. Results are means of 4 determinations, and are expressed relative to the original maximum contraction to sumatriptan obtained in Krebs containing 1.3mM CaCl2; vertical bars show s.e.mean. The 5-HT2 receptor antagonist, ritanserin (0.1 pM) was present throughout.

1008

10-7

(Table 1). This response was mimicked by the a2-adrenoceptor agonist, azepexole (10pM) but not by the al-adrenoceptor agonist, methoxamine (10 pM) (Table 1). In addition to its effects on PGE2-stimulated cyclic AMP formation, sumatriptan (10pM) also reduced both basal (51% reduction) and forskolin (5 pM)-stimulated (65% reduction) cyclic AMP formation (Table 2). Sumatriptan (1OpM)-induced inhibition of PGE2-stimulated cyclic AMP formation was antagonized by methiothepin (1 M), but not by spiperone, ondansetron, cyanopindolol, or mesulergine (all at 1 pM) or by metergoline at 0.1 M (Table 3). At these concentrations, none of the antagonists tested had any effect on PGE2-stimulated cyclic AMP formation in their

o

40-

2001I

10-3 10-4 10-5 Concentration (mol 1-1) dog isolated saphenous vein by sumatriptan of 4 Contraction Figure in the presence of verapamil. Initial cumulative concentration-effect curves to sumatriptan were established on all preparations. Each preparation was then exposed to vehicle (U) or to verapamil at 1 gM before repeating the (El), 3pM (V), 1OpM (0) or 30M (A) for of30min curves to sumatriptan. Results are means 4 determinations, and are expressed relative to the initial maximum response to sumatriptan; vertical bars show s.e.mean. The calcium concentration was 1.3 mM and ritanserin (0.1 pM) was continuously present throughout.

10-8

10-7

10-6

Table 1 Inhibition of prostaglandin E2 (PGE2)-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in rings of dog isolated saphenous vein Treatment

Cyclic AMP (pmol mg-' protein min

None PGE2 (5 pM) controls

PGE2 + sumatriptan (10pM)

PGE2 + 5-HT (10pM) PGE2 + 5-carboxamidotryptamine PGE2 + azepexole (1OpM) PGE2 + methoxamine (10pM) PGE2 + dideoxyadenosine (1 mM)

(10pM)

3.18 (±0.62) 6.87 (±1.00) 4.07 (±0.84)* 4.16 (+0.70)* 4.40 (±0.81)* 3.92 (+0.26)* 5.94 (±0.77) 5.03 (±0.45)*

Results are mean (± s.e.mean) of 3-6 determinations. Note that all of the agonists tested inhibited PGE2-stimulated cyclic AMP formation with the single exception of methoxamine. * Significantly different from PGE2 control (P < 0.05).

200 C 0

C

0

E E Co

C

605

120

80-

ECo 10-

lo-7

106--

04

1

Table 2 Effect of sumatriptan on basal, prostaglandin E2 (PGE2)-stimulated and forskolin-stimulated adenosine 3': 5'cyclic monophosphate (cyclic AMP) formation in rings of dog isolated saphenous vein

5)40 0

Treatment i0

i7 0

1o-3

10-5 10-6 104 Concentration (mol 1-1) Figure 3 Contraction of dog isolated saphenous vein by 5hydroxytryptamine (5-HT) in the presence of verapamil. An initial cumulative concentration-effect curve to 5-HT was established in all preparations. Each preparation was then exposed to vehicle (U) or verapamil at 3pM (O), 10pM (A) or 30OM (V) for 30min before repeating the concentration-effect curve to 5-HT. Results are means of 4 determinations and are expressed relative to the initial maximum response to 5-HT; vertical bars show s.e.mean. The Krebs solution contained 1.3 mM CaCl2 and 0.1 pM ritanserin throughout.

Basal control Basal + sumatriptan (10pM) PGE2 (5pM) control PGE2 (5pM) + sumatriptan (10puM) Forskolin (5 pM) control Forskolin (5 pM) + sumatriptan (10pM)

Cyclic AMP (pmol mg 1 protein min -) 3.18 (±0.62) 1.56 (±0.47)* 6.70 (±0.47) 3.98 (±0.44)* 14.58 (±1.01) 5.15 (±0.96)*

Results are mean (± s.e. mean) of 4-6 determinations. * Significantly different from the corresponding control (P < 0.05).

')

606

M.J. SUMNER et al. Table 3 Effect of sumatriptan on prostaglandin E2

a

lOr-

(PGE2)-stimulated adenosine 3': 5'-cyclic monophosphate (cyclic AMP) formation in rings of dog isolated saphenous vein in the absence and presence of various 5hydroxytryptamine (5-HT) receptor antagonists Treatment

Basal control Basal + sumatriptan (1OuM) PGE2 control PGE2 + sumatriptan (10pM) PGE2 + sumatriptan + spiperone (1 FM) PGE2 + sumatriptan + ondansetron (1 M) PGE2 + sumatriptan + cyanopindolol (1 pM) PGE2 + sumatriptan + mesulergine (1 FM) PGE2 + sumatriptan + metergoline (0.1 pM) PGE2 + sumatriptan + methiothepin (1 pM)

._ ._

E0

Cyclic AMP (pmol mg 1 protein min -1)

EQ E

3.18 (±0.62) 1.56 (±0.30) 6.70 (±0.47) 3.98 (±0.44)*

5

E

C.)a

T

0-

4.10 (±0.26)* ._

3.60 (± 0.42)*

o

C

3.08 (±0.34)*

I

5-HT

C

5-HT

C

5-HT

3.11 (±0.39)*

3.65 (±0.44)* 10

6.42 (±0.98)

Results are mean (± s.e.mean) of 6 determinations. All experiments were performed in the presence of PGE2 (5pM), with the exception of the basal determinations in the absence or presence of sumatriptan. * Significantly different from PGE2 control, P < 0.05. t At concentrations above 0.1 pM, metergoline displayed

agonist activity (see results).

0oI

8

E E oC C

6

.2

4

E .'

b

1

0

2

own right. However, at higher concentrations, metergoline (1 PM) and methysergide (1oPM) also inhibited PGE2-stimulated cyclic AMP formation, although only the effect of metergoline proved to be significant (mean inhibition of PGE2-stimulated cyclic AMP formation was 23 + 5% for metergoline (1pM) and 23 + 15% for methysergide (1OpM) compared to 33 + 3% for sumatriptan (10 gM), n = 4). The effects of these antagonists on the inhibition of basal cyclic AMP formation produced by sumatriptan were not examined. Incubation of rings of dog saphenous vein in Ca2 + -free Krebs solution containing 1 mm EGTA or in Krebs solution containing 1.3 mm Ca2 + and 1 pM verapamil did not affect the inhibition of PGE2-stimulated cyclic AMP formation produced either by 5-HT (Figure 5a) or by sumatriptan (Figure 5b). A directly-acting inhibitor of adenylyl cyclase, 2',3'-dideoxyadenosine (Haslam et al., 1978) also inhibited (only significantly at 1 mM) PGE2-stimulated cyclic AMP formation (Table 1), but unlike sumatriptan, did not evoke contraction of strips of dog isolated saphenous vein (results not shown).

Discussion The intracellular mechanism of 5-HT1-like receptor-mediated smooth muscle contraction in the dog isolated saphenous vein is not known (Apperley et al., 1980; Humphrey et al., 1988). In this study we have demonstrated that sustained smooth muscle contraction arising through activation of 5-HT1-like receptors by either 5-HT or sumatriptan is dependent upon extracellular calcium, and is largely attenuated, but not abolished, by verapamil, a blocker of voltage-dependent calcium channels (Fleckenstein 1977). Interestingly, the contractile effects of high concentrations of 5-HT, which arise indirectly through noradrenaline release and activation of postjunctional a-adrenoceptors (Humphrey, 1978), probably of the a1-subtype (Mathews et al., 1984), were far less sensitive to either removal of extracellular calcium or to verapamil. These results would argue against a non-specific effect of verapamil, and are in keeping with those obtained with selective adrenoceptor agonists in this tissue (Mathews et al., 1984;

o0

0

C S C S Figure 5 Effect of 5-hydroxytryptamine (5-HT) (a) and sumatriptan (b) on prostaglandin E2 (PGE2)-stimulated adenosine 3': 5'-cyclic monophosphate (cyclic AMP) formation in dog isolated saphenous vein in the absence of calcium or in the presence of verapamil. Rings of dog isolated saphenous vein were incubated for 30 min in Krebs solution containing 100pM 3-isobutyl-1-methylxanthine (IBMX) and 1.3mm CaCl2, in the absence (open columns) or presence of 1 UM verapamil (cross hatched columns), or in calcium-free Krebs containing 100pM IBMX and 1 mM EGTA (solid columns). Rings were then exposed for 2 min to PGE2 (5pjM) and either vehicle (C) or 10pM 5-HT (a) or 10pM sumatriptan (denoted S in b) before extracting and assaying the protein and cyclic AMP content of each ring. Results are means of 4 (5-HT) or 5 (sumatriptan) determinations; vertical bars show s.e. mean. * P < 0.05 relative to the appropriate controls. C

Bulbring & Tomita, 1987; Docherty, 1989) and are compatible with a major role for intracellular calcium mobilization in a1-adrenoceptor-mediated responses (Fain & Garcia-Sainz, 1980). Furthermore, these observations would also suggest that 5-HT1-like and cxl-adrenoceptor-mediated smooth muscle contraction arise largely through different mechanisms, involving the influx of extracellular calcium (largely through voltage-dependent channels) or the mobilization of intracellular calcium respectively. It is recognised, however, that the initial contractile response to 5-HT1-like receptor activation may be more dependent upon mobilisation of intracellular calcium stores, with the subsequent sustained phase of contraction being supported by an influx of extracellular calcium. Although this possibility was not specifically addressed in this study, there was no evidence for any contraction to 5-HT or sumatriptan in nominally calcium-free Krebs solution, irrespective of whether cumulative concentration-effect curves (see Results) or single concentrations (results not shown) were examined. As well as causing smooth muscle contraction, 5-HT and sumatriptan also reduced PGE2-stimulated cyclic AMP formation, possibly due to an inhibition of adenylyl cyclase. Since agents which increase cyclic AMP concentrations in this

5-HT1 RECEPTOR-INDUCED SMOOTH MUSCLE CONTRACTION

tissue also cause smooth muscle relaxation (Kikkawa et al., 1986), it is conceivable that reducing cyclic AMP levels could lead, at least in part, to smooth muscle contraction. In keeping with this, the a2-adrenoceptor agonist, azepexole (B-HT933), also inhibited cyclic AMP formation (this study) and caused smooth contraction (Ruffolo & Zeid, 1985; Rhodes & Waterfall, 1987) in this tissue. A number of adenosine analogues have been shown to inhibit adenylyl cyclase through a direct action at the so-called 'P-site' on the enzyme (Haslam et al., 1978). One such compound, 2',3'-dideoxyadenosine, although significantly reducing cyclic AMP levels (Table 1), failed to elicit smooth muscle contraction. It is possible that cyclic AMP concentrations need to be reduced beyond a critical level in order to evoke contraction. Indeed, the 5-HT1-like receptor agonists, which both caused contraction of dog saphenous vein, also elicited a greater reduction in cyclic AMP levels than did dideoxyadenosine. Nevertheless, this result would suggest that a reduction in cyclic AMP levels per se is not likely to be directly responsible for smooth muscle contraction. An alternative mechanism might be one in which a rise in intracellular calcium levels brings about both smooth muscle contraction and a fall in cyclic AMP levels. In this regard, calcium can clearly modulate cyclic AMP formation and breakdown (Rasmussen & Barrett, 1984; Resink et al., 1986). However, such a mechanism appears unlikely in view of the lack of effect of either reducing the extracellular calcium concentration, or of verapamil, on 5-HT1-like receptor-mediated reductions in cyclic AMP concentrations, compared with the marked effect that was observed on 5-HT1-like receptormediated smooth muscle contraction. Any mechanism which attempts to account for 5-HT1-like receptor-mediated smooth muscle contraction and inhibition of cyclic AMP formation must accommodate the experimental observations that contraction: (1) is totally dependent upon extracellular calcium, a large component of which appears to enter the cell via voltage-dependent calcium channels, (2) is clearly different from a-adrenoceptor-mediated contraction

607

(which probably involves a1-receptor-mediated mobilization of intracellular calcium stores) and (3) is accompanied by a concomitant inhibition of adenylyl cyclase which appears to be an independent event, despite being evoked via the same receptor. A mechanism which could account for all these observations is one in which 5-HT1-like receptor-mediated inhibition of adenylyl cyclase and activation of a cationic channel occur independently. The ensuing cellular depolarisation would open voltage-dependent calcium channels thereby propagating the calcium influx, leading to smooth muscle contractions. The latter response may be facilitated by the accompanying fall in cyclic AMP levels (Rasmussen & Barrett, 1984). Sumatriptan has been reported to act as an agonist at brain 5-HTID receptors (Schoeffter & Hoyer, 1989). These brain receptors are similar to dog saphenous vein 5-HT1-like receptors, in that both appear to be negatively coupled to adenylyl cyclase and both are sensitive to 5-HT, 5-CT and sumatriptan but not to spiperone (Waeber et al., 1988; Schoeffter & Hoyer, 1989; Sumner & Humphrey, 1990; this study). However, metergoline appears to differentiate between brain 5-HTID receptors, for which it shows a high affinity (Waeber et al., 1988), and the dog saphenous vein 5-HT1-like receptor, at which it appears to act as a weak partial agonist (Perren et al., 1991). The results of this study confirm the weak partial agonist activity of metergoline at the dog saphenous vein 5-HT1-like receptor. It is of interest to note, however, that studies using a different central 5-HTlD receptor model (Middlemiss et al., 1988) found methysergide and metergoline to be weak antagonists or partial agonists. These data are in keeping with those obtained in this study, and support the notion that brain 5-HTID receptors and the dog saphenous vein 5-HT1-like receptor may be similar. Nevertheless, the definitive characterisation of these two important 5-HT, receptor sites, both of which are negatively coupled to adenylyl cyclase, must await the identification of specific and selective receptor antagonists.

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Evidence for two types of excitatory receptor for 5hydroxytryptamine in dog isolated vasculature. Br. J. Pharmacol., 68,215-224. BOYER, J.L., CARDENAS, C., POSADAS, C. & GARCIA-SAINZ, J.A. (1983). Pertussis toxin induces tachycardia and impairs the increase in blood pressure produced by alpha2-adrenergic agonists. Life Sci., 33, 2627-2633. BOLBRING, E. & TOMITA, T. (1987). Catecholamine action on smooth muscle. Pharmacol. Rev., 39, 49-96. DOCHERTY, J.R. (1989). The pharmacology of a1- and a2-adrenoceptors: Evidence for and against a further subdivision. Pharmacol. Ther., 44, 241-284. FAIN, J.N. & GARCIA-SAINZ, J.A. (1980). Minireview. Role of phosphatidylinositol turnover in alpha, and of adenylate cyclase inhibition in alpha2 effects of catecholamines. Life Sci., 26, 1183-1194. FLECKENSTEIN, A. (1977). Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Annu. Rev. Pharmacol. Toxicol., 17, 149-166. HASLAM, R.J., DAVIDSON, M.M.L. & DESJARDINS, J.V. (1978). Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP. Biochem. J., 176, 83-95. HUMPHREY, P.P.A. (1978). The effects of a-adrenoceptor antagonists on contractile responses to 5-hydroxytryptamine in dog saphenous vein. Br. J. Pharmacol., 63, 671-675. HUMPHREY, P.P.A., FENIUK, W., PERREN, MJ., CONNOR, H.E.,

OXFORD, A.W., COATES, I.H. & BUTINA, D. (1988). GR43175, a selective agonist for the 5-HT1-like receptor in dog isolated saphenous vein. Br. J. Pharmacol., 94, 1123-1132. KIKKAWA, F., FURUTA, T., ISHIKAWA, N. & SHIGEI, T. (1986). Different types of relationship between f,-adrenergic relaxation and activation of cyclic AMP-dependent protein kinase in canine

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