1 In the present study, depletion of internal Ca2+ stores sensitive to noradrenaline (1 jiM) in rat aorta, is the signal for the entry of extracellular Ca2", not only to refill the stores but also, in our experimental conditions, to activate the contractile proteins. This induces an increase in the resting tone that constitutes, the first functional evidence of this Ca2" entry.2 The fact that methoxamine (100 gM) reproduces the same processes as noradrenaline but clonidine (1 gM) does not, indicates that a,-adrenoceptor activation is related to the increase in the resting tone observed after depletion of adrenoceptor-sensitive internal Ca2+-stores.3 Benoxathian and WB 4101 (alA-and a,D-adrenoceptor antagonists) selectively inhibit, in a concentration-dependent manner, this mechanical response observed in absence of the agonist, which suggests that these agents can act as inverse agonists and provide a functional model for studying this phenomenon. Since chloroethylclonidine (100 gM) has no effect on this response, the participation of cxl]3-adrenoceptors can be ruled out.4 Contractile responses to noradrenaline (1 gM) in Ca2+-free medium were selectively blocked by chloroethylclonidine. This suggests that the response to noradrenaline in Ca2+-free medium mainly depends on the activation of the alB-adrenoceptor subtype.

1 In the present study, the properties of glaucine (an aporphine structurally related to papaverine) were compared with those of papaverine, diltiazem, nifedipine and prazosin. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of glaucine at calcium channel binding sites of x-adrenoceptors, by use of [3H]-( + )-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The effects of glaucine on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2 Contraction evoked by noradrenaline (1 gLM) or depolarizing solution (60 mM KCl) were inhibited in a concentration-dependent manner by all the compounds tested. As expected, prazosin showed a greater selectivity of action on NA-induced contraction, whereas nifedipine and diltiazem appeared more potent on KCl-induced contraction. Glaucine had a greater potency on the contraction elicited by noradrenaline whereas papaverine acted non specifically.3 In Ca2'-free solution, prazosin (0.1 pM) and glaucine (0.1 mM) inhibited the contraction evoked by NA; diltiazem (0.1 mM) diminished this contraction whereas nifedipine (1 pM) had no effect. Preincubation of tissues with glaucine, diltiazem, nifedipine and prazosin did not modify the contractile response induced by caffeine. In contrast, papaverine (0.1 mM) significantly inhibited the contractions evoked by NA or caffeine in Ca2"-free medium. 5 This study confirms the presence of four phosphodiesterase (PDE) activities in bovine aorta: a calmodulin-activated PDE (CaM-PDE type I) which hydrolyzed preferentially guanosine 3':5'-cyclic monophosphate (cyclic GMP); a cyclic GMP selective form (cGMP-PDE type V); and two low Km adenosine 3':5'-cyclic monophosphate (cyclic AMP) PDEs that are insensitive to the stimulatory effect of CaM, one of which was inhibited by cyclic GMP (CGI-PDE, type III) and the other by rolipram (cAMP-PDE, type IV). Glaucine selectively inhibits one of the two forms of Ca2+-independent low Km cAMP-PDE, the type IV. In contrast, papaverine exerts a non-selective inhibitory effect upon all PDE forms.6 The present work provides evidence that glaucine, a benzyltetrahydroisoquinoline alkaloid, has interesting properties as an al-adrenoceptor antagonist, calcium entry blocker (through the benzothiazepine recognition site in the calcium channel) and as a selective inhibitor of the rolipram-sensitive cAMP-PDE, type IV PDE.

Figure 3 amended [23 January 2015] to correct error in text. BACKGROUND AND PURPOSETo analyse the relative contribution of b1-, b2-and b3-adrenoceptors (Adrb) to vasodilatation in conductance and resistance vessels, assessing the role of cAMP and/or NO/cGMP signalling pathways. EXPERIMENTAL APPROACHRat mesenteric resistance artery (MRA) and aorta were used to analyse the Adrb expression by real-time-PCR and immunohistochemistry, and for the pharmacological characterization of Adrb-mediated activity by wire myography and tissue nucleotide accumulation. KEY RESULTSThe mRNAs and protein for all Adrb were identified in endothelium and/or smooth muscle cells (SMCs) in both vessels. In MRA, Adrb1 signalled through cAMP, Adrb3 through both cAMP and cGMP, but Adrb2, did not activate nucleotide formation; isoprenaline relaxation was inhibited by propranolol (b1, b2), CGP20712A (b1), and SQ22536 (adenylyl cyclase inhibitor), but not by ICI118,551 (b2), SR59230A (b3), ODQ (soluble guanylyl cyclase inhibitor), L-NAME or endothelium removal. In aorta, Adrb1 signalled through cAMP, while b2-and b3-subtypes through cGMP; isoprenaline relaxation was inhibited by propranolol, ICI118,551, ODQ, L-NAME, and to a lesser extent, by endothelium removal. CL316243 (b3-agonist) relaxed aorta, but not MRA. CONCLUSION AND IMPLICATIONDespite all three Adrb subtypes being found in both vessels, Adrb1, located in SMCs and acting through the adenylyl cyclase/cAMP pathway, are primarily responsible for vasodilatation in MRA. However, Adrb-mediated vasodilatation in aorta is driven by endothelial Adrb2 and Adrb3, but also by the Adrb2 present in SMCs, and is coupled to the NO/cGMP pathway. These results could help to understand the different physiological roles played by Adrb signalling in regulating conductance and resistance vessels.

Noradrenaline (NA) 1 microM and caffeine (CAF) 10 mM induce a contractile response in isolated rat aorta maintained at 37 degrees C either in the presence or absence of extracellular calcium. In Ca-free media the contractile response was reduced and contractile activity of CAF only occurred at 25 degrees C. NA induced a biphasic response in Ca-free medium, with a fast phasic contraction followed by a smaller more sustained contraction. The response induced by CAF consisted of a fast transient contraction which returned to a level below the resting tone. After washing, further addition of NA or CAF evoked no increase in smooth muscle tension. The influence of Mg-depletion in the extracellular medium on the contractile responses induced by NA and CAF in Ca-free medium was determined: similar response to NA or CAF in media with or without Mg after 2 min loading were observed, but after 15 min loading in Mg, Ca-free solution, the responses to NA or CAF were significantly higher than after incubation in Ca-free medium containing Mg. Differences were observed when contractile response to NA was elicited after the refilling process by loading the aortic strip in Ca-containing Mg-free solution. In this case, instead of recovering the magnitude of contraction there was a significant decrease. The existence of two independent intracellular Ca-pools releasable by NA, one of them also sensitive to CAF, is postulated. The refill of the Ca-store specific to NA is dependent on the presence of Mg in the extracellular medium. In contrast, the refill of the common Ca-pool releasable by NA and CAF is independent of the extracellular Mg, but its spontaneous emptying is temperature-, and Mg-dependent. This suggests the intermediacy of an enzymatic process to extrude the Ca-content of this store to the extracellular space.

1 The role of a constitutively active population of a 1D -adrenoceptors was analysed in arteries obtained from spontaneously hypertensive rats (SHR) and controls (WKY) divided into three groups: young prehypertensive, adult hypertensive, and adult animals chronically treated with captopril (50 mg kg 71 per day orally) in order to prevent the hypertensive state. 2 In adult SHR, a signi®cant increase in BMY 7378 potency (not in prazosin potency) was observed in aorta, mesenteric artery, and the ®rst and second branches of the small mesenteric arteries with respect to WKY rats. This di erence was not observed in iliac and tail arteries, which suggests an increased functional role of a 1D -adrenoceptors only in some vessels of SHR. 3 The increase in the resting tone (IRT) observed in absence of agonist, inhibited by BMY 7378, that represents the constitutively active population of a 1D -adrenoceptors, was also signi®cantly greater in aorta and mesenteric artery from adult SHR. 4 In young and captopril treated adult animals, no di erences between strains with respect to BMY 7378 potency, or IRT were observed. 5 The increase in the functional role of a 1D -adrenoceptors and their constitutive activity observed in hypertension is prevented by captopril treatment. The pathological consequence of this change is the slower rate of recovery of the basal tone after removal of an adrenergic stimulus, observed in vessels from hypertensive animals that had shown an increase in the functionality of constitutively active a 1D -adrenoceptors. This change was not observed in prehypertensive or captopril treated animals. IRT, increase in the resting tone; NA, noradrenaline; SHR, spontaneously hypertensive rats; SMA-1 and SMA-2, ®rst and second small mesenteric arterial branches; WKY, Wistar Kyoto rats