Chris R. Triggle

The effect of the calcium channel agonist, Bay K-8644 on human vascular smooth muscle

Bay K-8644, a calcium channel activator, caused a dose-dependent (ED50 = 12.8 nM) elevation of tone in the human umbilical artery. The response to Bay K-8644 was dependent upon extracellular calcium (Ca2+), competitively antagonized by the channel antagonist, nifedipine, and, in addition, the sensitivity to Bay K-8644 was increased by, but not dependent upon, elevating extracellular potassium. Bay K-8644 competitively displaced, IC50 = 1.8 nM, specific [3H]nitrendipine binding to a microsome fraction derived from the umbilical artery, suggesting that the Ca2+ channel activator and the structurally related dihydropyridine antagonists bind to a single site to affect Ca2+ channel function in human vascular smooth muscle.

Pharmacological profile of the 5-hydroxytryptamine receptor that mediates relaxation of rat oesophageal smooth muscle

1. The pharmacological profile of the inhibitory 5-hydroxytryptamine (5-HT) receptor in rat oesophageal smooth muscle has been characterized by means of a series of agonists active at 5-HT1-, 5-HT2-, 5-HT3- and 5-HT4-receptor sites, and a broad range of antagonists. The possible involvement of cyclic nucleotides in the 5-HT response was also examined. 2. Under conditions of tone induced by muscarinic receptor activation, the upper two-thirds (proximal segment) of the oesophageal smooth muscle tunic was more sensitive to the inhibitory effects of 5-HT receptor agonists when compared with the distal region. 3. The inhibitory response to 5-HT was blocked by MDL 72222 (5-HT3 antagonist) and ICS 205-930 (5-HT3/5-HT4 antagonist) but not by antagonists active at 5-HT1- or 5-HT2-receptors. 4. The phosphodiesterase inhibitor, 3-isobutyl-methyl-xanthine (IBMX) enhanced oesophageal smooth muscle inhibitory response to 5-HT, isoprenaline and forskolin, but not that elicited by the potassium channel opener, BRL 34915. 5. 5-HT increased tissue cyclic AMP content over basal levels in proximal and distal segments of oesophageal smooth muscle. However, 5-HT had no significant effect on basal cyclic GMP levels in both segments. 6. We conclude that the inhibitory 5-HT receptor in rat oesophageal smooth muscle may represent a high affinity subtype which is sensitive to 5-HT3/5-HT4 antagonists and is coupled to the cyclic AMP pathway.

α-Adrenoceptor activation of polyphosphoinositide hydrolysis in the rat tail artery

Abstract α-Adrenoceptor coupling to polyphosphoinositide (PI) hydrolysis was studied in the rat tail artery. Inositol phosphate (IP) accumulation was stimulated by the non-selective α-adrenoceptor agonist norepinephrine and the α 1 -adrenoceptor agonist phenylephrine. This stimulation was relatively dependent on extracellular Ca 2+ and enhanced markedly in the presence of LiCl. In addition, norepinephrine- and phenylephrine-stimulated IP accumulation was relatively sensitive to blockade by prazosin, compared to rauwolscine. The putative α 2 -adrenoceptor agonist UK 14304 also stimulated PI breakdown in a concentration-dependent manner, although this stimulation did not reach equilibrium at up to 10 mM and was relatively sensitive to prazosin, compared to rauwolscine, over the lower agonist concentrations. NaF stimulated IP accumulation independently of α-adrenoceptor activation. PI breakdown by α-adrenoceptor agonists and NaF was attenuated by N -ethylmaleimide but not pertussis toxin treatment. In addition, dithiothreitol blocked NaF-stimulated, but not α-adrenoceptor-mediated, PI breakdown. These results suggest the coupling of α 1 -adrenoceptor, via phospholipase C, to PI hydrolysis in the rat tail artery. This study also provides evidence for the involvement of one or more non-G i -like G-protein(s) in the signal transduction process.

Alpha adrenoceptor sites in vascular smooth muscle: Differentiation by selective antagonist binding

The properties of alpha adrenoceptors in rat-tail artery membranes were studied using tritiated ligands that are selective for the alpha 1 and alpha 2 subtypes. High-affinity saturable binding was obtained for the alpha 1 antagonist prazosin yielding a Bmax of 144 +/- 31.6 fmol/mg protein (mean +/- SEM, N = 3) and a Kd of 0.17 +/- 0.04 nM, and also for the alpha 2 antagonist rauwolscine which yielded a Bmax of 141.3 +/- 19.3 fmol/mg protein and a Kd of 1.57 +/- 0.32 nM. The [3H]prazosin-labelled sites displayed a pharmacological profile characteristic of an alpha 1 adrenoceptor, whereas the [3H]rauwolscine-labelled sites exhibited the expected alpha 2 adrenoceptor profile. Agonist affinity for [3H]rauwolscine sites was reduced by Gpp(NH)p and Na+, and the effects appeared synergistic for adrenaline, but non-interactive for UK-14304. Agonist interaction with [3H]prazosin sites in the rat-tail artery was also regulated by Gpp(NH)p and Na+, although clearly in a qualitatively and quantitatively different manner from the [3H]rauwolscine sites. These results suggest that distinct binding sites for [3H]prazosin and [3H]rauwolscine could be differentiated with antagonist ligands. These distinct antagonist recognition sites demonstrate the pharmacological profile expected for alpha 1 and alpha 2 adrenoceptors, and the quantitatively differing abilities of Na+ and Gpp(NH)p to regulate agonist interactions with these sites are suggestive, but do not necessarily prove, that different G proteins may be involved in this regulation.

Nitric oxide, a possible mediator of 1,4‐dihydropyridine‐induced photorelaxation of vascular smooth muscle

1 In rat aortic tissues pre‐contracted with phenylephrine, certain 1,4‐dihydropyridines (DHPs) such as Bay K 8644 (0.1 μm), PN 202791 (1 μm), RK 30 (1 μm), NI 104 (1 μm) and NI 105 (1 μm) enhanced photoactivated relaxations (photorelaxation or PR) whereas NI 72, NI 85, NI 99, NI 102, amlodipine, felodipine, nifedipine and nimodipine were inactive. 2 The PR inducing effects of Bay K 8644 were mimicked by the diabetogenic agent, streptozotocin (STZ). 3 Solutions of Bay K 8644 which had been irradiated for various periods of time initiated light independent transient relaxations followed by contractile responses in aortic tissue partially contracted with phenylephrine. With exposure times to light of 30 to 120 min, the intensity of the relaxation response to irradiated Bay K 8644 increased from 26±3.3 to 71±3.7% of the maximum contractile response to phenylephrine (n = 5). Conversely the contractile responses decreased, from 84.2±4.1 to 19.8±10.4% of the maximum contractile response to phenylephrine (n = 5). 4 Superoxide ions, generated by incubation of xanthine (2 mM) plus xanthine oxidase (10 mu ml−1) in physiological saline solution (PSS) NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 12.5 and glucose 11.1 (mM) for 1 h, reduced the PR induced by DHPs, STZ, and also NO‐induced relaxations of rat aortic preparations. 5 Direct measurements of NO indicate that, following exposure to a polychromatic light source, equimolar concentrations (0.1 mM) of the DHP compounds that enhance PR, as well as STZ, photodegrade to release NO (25±2‐40.3±5.9 nmol min−1, n = 6). 6 Structure‐activity studies indicate that a nitro group at the −3 position of the dihydropyridine ring is essential for DHPs to support PR. 7 These data suggest that the photodegradation of DHPs and STZ leading to the release of NO provides the primary cellular process underlying the PR response.

A comparative study of endothelin and sarafotoxin action in vascular and non-vascular smooth muscle.

The effects of endothelin and sarafotoxin on smooth muscle tone have been examined in the rat aorta and anococcygeus muscle and their actions compared to those of norepinephrine. The contractions elicited by endothelin and sarafotoxin (10 nM), or norepinephrine (1 ?M) were approximately equieffective in terms of tension development and correspond to EC(50) values and these concentrations were thus used throughout the study. In calcium-free Krebs the three agonists generated approximately similar levels of tone in the aorta and the anococcygeus corresponding to 20 and 5% of the maximum response, respectively. Nifedipine, 10 ?M, significantly inhibited responses to endothelin and norepinephrine in the aorta but only norepinephrine in anococcygeus; the responses to sarafotoxin were however not significantly affected in either tissue. A combination of 10 ?M ryanodine and nifedipine caused near complete inhibition of the response to endothelin in the aorta and also significantly reduced the response to both endothelin and norepinephrine in the anococcygeus. The lipoxgenase inhibitor, nordihydroguaiaretic acid, inhibited the response to endothelin in the aorta and endothelin and norepinephrine in the anococcygeus muscle. The cyclooxygenase inhibitor, indomethacin, however, had no effect on the responses to any of the three agonists in either the aorta or anococcygeus. At concentrations greater than 30 nM both endothelin and sarafotoxin induced myogenic activity in normally quiescent anococcygeus muscle. As determined by the loss of myogenic activity the tissues recovered more rapidly from sarafotoxin than endothelin with complete recovery apparent after 2.62 +/- 0.85 and 5.22 +/- 0.06 h respectively. Omitting Ca(2+) from the Krebs solution reduced recovery times to 1.62 +/- 0.2 and 2.4 +/- 0.51 h respectively. Overall the results suggest that endothelin and sarafotoxin activate different cell signaling systems to differing extents in rat aorta versus anococcygeus suggesting that the membrane receptors mediating the responses to endothelin and sarafotoxin are not necessarily identical.

Similarity of relaxations evoked by BRL 34915, pinacidil and field-stimulation in rat oesophageal tunica muscularis mucosae

1 In the rat oesophageal tunica muscularis mucosae (TMM) the potassium channel openers, BRL 34915 and pinacidil, raised the threshold for concentration‐dependent K+ contractions, suppressed contractions evoked by field stimulation of the TMM in the presence of tetrodotoxin (TTX) and tetraethylammonium (TEA), and relaxed tonic contractions resulting from muscarinic cholinoceptor stimulation. 2 BRL 34915 and both (+)− and (−)‐pinacidil increased 86Rb efflux from tracer‐loaded tissues; nifedipine abolished this effect. 3 Relaxations produced by potassium channel openers were inhibited by a temperature drop from 37°C to 26.5°C, an increase in extracellular K+ concentration to 64 mm, and treatment with the calcium channel antagonist, nifedipine. The same treatments also blocked field stimulation‐evoked TTX‐insensitive relaxations. 4 It is concluded that field stimulation of rat oesophageal smooth muscle in the presence of cholinoceptor‐induced tone results in an increase in K+ permeability that is directly or indirectly coupled to Ca2+ influx through potential‐operated channels.

Benextramine and nifedipine distinguish between sub-classes of α1-adrenoceptors

Abstract The effects of benextramine and nifedipine were examined on the dosediastolic pressure response to methoxamine in pithed normotensive rats. Benextramine (3, 6 and 12 mg/Kg) displaced the dose-response curve to methoxamine to the right. Maximum response was reduced after the administration of 12 mg/Kg benextramine. Nifedipine (0.1 and 0.3 mg/Kg) also caused the dose-response curve to methoxamine to be displaced to the right with reduction in maximum response. Nifedipine effects were additive with an increase in the EC50 values as well as reduction in the maximum response after pretreatment with benextramine (3 and 6 mg/Kg). However, at the highest dose of benextramine the effects of nifedipine were diminished and no longer apparent. It is concluded that benextramine may have alkylated a nifedipine sensitive site on the α1-adrenoceptors.

Inhibition of field stimulation-evoked relaxations in rat oesophageal smooth muscle by the calcium antagonist PN 200–110

1 The inhibitory effects of the 1,4‐dihydropyridine calcium channel antagonist, PN 200–110 (isradipine), on field stimulation‐evoked tetrodotoxin (TTX)‐sensitive and‐insensitive relaxations were studied in rat oesophageal smooth muscle of the tunica muscularis mucosae. 2 The TTX‐insensitive relaxation was inhibited by PN 200–110 in a stereoselective manner with the (+)−(S)‐isomer displaying a 1000 fold greater inhibitory potency than the (—)−(R) isomer. A similar potency was noted for inhibition of high K+‐evoked contractions. 3 TTX‐sensitive relaxations evoked by field stimulation and contractions elicited by the muscarinic cholinoceptor agonist, cis‐2‐methyl‐4‐dimethylamino‐methyl‐1,3‐dioxolane methiodide (cis‐dioxolane) were considerably less sensitive to inhibition by PN 200–110, although, again, stereoselectivity for PN 200–110 was apparent. 4 Pretreatment with (+)−(S)‐PN 200–110 resulted in a non‐competitive displacement of the Ca2+ concentration‐response curves obtained in the presence of either isotonic 50 mm KCl or cis‐dioxolane. The effect of K+ was 10 fold more sensitive than that of cis‐dioxolane. 5 The potency rank orders for inhibition of TTX‐insensitive field stimulation‐evoked relaxations and K+‐mediated contractions in a series of calcium channel antagonists were closely correlated; (+)−(S)‐PN 200–110 showing highest potency followed by nifedipine, verapamil, diltiazem, (—)−(R)‐PN 200–110. 6 It is concluded that TTX‐insensitive relaxations are dependent upon an influx of extracellular Ca2+ through potential‐operated calcium channels.

Calcium antagonizes the magnesium-induced high affinity state of the hepatic vasopressin receptor for the agonist interaction

1 The present study describes the role of Ca2+ in the regulation of the hepatic vasopressin V1 receptor. With low concentrations of Ca2+, there was a small increase in [3H]‐arginine vasopressin ([3H]‐AVP) binding, but above 10 mm, Ca2+ decreased the binding of this agonist. In contrast, low concentrations of Mg2+ were associated with a dramatic concentration‐dependent increase in [3H]‐AVP binding, reaching a maximal effect of 650% above control at concentrations ranging between 1–5 mm. At higher concentrations of Mg2+, the stimulatory effect of this cation was less pronounced, falling to 210% of control at 100 mm Mg2+. Strikingly, Ca2+‐inhibited the stimulatory effect of Mg2+ in a concentration‐dependent fashion. 2 Saturation binding data revealed that Ca2+ (2 to 10 mm) per se promotes the high affinity conformation of the V1 receptor for the agonist binding with the KD decreased from a control value of 2.3 nm to 0.5 nm in the presence of 10 mm Ca2+. This effect was attenuated with an increase in Ca2+ above 10 mm. With an increase in Ca2+ to 20 mm, however, the Bmax for [3H]‐AVP binding was decreased. Ca2+ also decreased the high affinity/high capacity state (KD 100 pm) of the receptor induced by 1 mm Mg2+ for agonist interaction. 3 [3H]‐V1 antagonist binding was inhibited by both Ca2+ and Mg2+. The IC50 values (mean ± s.e.mean) for Ca2+ and Mg2+ were 32 ± 8 and 53 ± 9 mm respectively. Maximal inhibition achieved at 100 mm was 29% for Ca2+ and 42% for Mg2+. Both cations decreased the affinity and increased the capacity of the V1 receptor for the antagonist. 4 The results suggest that the divalent metal ion binding site(s) modulated by Mg2+ is also accessible to Ca2+. Although Ca2+ opposes the powerful stimulatory effects of Mg2+ on agonist binding, the effects of Ca2+ and Mg2+ on the Bmax of [3H]‐AVP binding were different, suggesting that the divalent cations may bind to two different sites, thereby regulating the affinity and the capacity characteristics of the V1 receptor.