Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5'-(fly-imidoltriphosphate (p[NH]ppG), followed by treatment with GTP and washing at 20C. Activation (C-.C*) proceeded in a timeand temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 370C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 370C. Over the range 6-240C, activation by pretreatment with p[NH]ppG followed simple Michaelis-Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased Km for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (370C) was moderately endergonic (AHO -8kJ.mol-') and accompanied by an increase in entropy (ASO 146 J * mol-h * K-1). The 6-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P > 0.05) effect on the Km at every temperature. This result suggested that formation of the receptor-hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the fadrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.

Aortic smooth muscle cells from 3, 12 and 24 mo old rats were grown in cell culture. All of the cultures produced angiotensinogen, but cultures from 24 mo old rats produced significantly more (p less than 0.01). These results suggest that angiotensinogen is synthesized in arterial smooth muscle. Furthermore, when the activity of the plasma renin system decreases with advancing age, increased activity of a local angiotensin system may contribute to the regulation of vascular tone.

It was the purpose of this study to determine whether acute increases of endogenous plasma renin induced by administration of anesthetics cause simultaneous increases of the enzyme in aortic tissue of the rat. Administration of CO2 did not alter plasma or tissue renin. Ether, Innovar and Nembutal increased active plasma renin and had variable effects on the inactive form of the enzyme. Only Innovar, a combination of droperidol and fentanyl, increased aortic renin. The active component of Innovar was shown to be droperidol, which also increased aortic renin in 24 hour nephrectomized animals. The increase of aortic renin was, therefore, independent of changes of circulating active or inactive renin. The increase of tissue renin following droperidol was rapid, suggesting activation of an inactive tissue renin.

The estrogen sensitivity of cells cultured from the rat myometrium was studied by growing the cells in the absence or presence of 1 nM 17 beta-estradiol. Following a time lag of approximately 10 days, exposure to estrogen resulted in increased incorporation of radiothymidine by the cells. Estrogen treatment also decreased isoproterenol-dependent and GTP-dependent adenylate cyclase activity, but had no effect on basal activity. These cultured cells have been shown previously to have some properties of uterine smooth muscle. The effects estrogen has in vitro, therefore, may reflect important properties in vivo that account for the mechanism by which the sex steroid decreases the sensitivity of the myometrium to isoproterenol.

To compare the inhibition of uterine contractility mediated by β-adrenoceptors and 5-HT7 receptors, the effects of catecholamines and 5-HT on spontaneous contractions were examined in longitudinal and circular muscles isolated from three different regions (cornu, corpus and cervix) of the non-pregnant proestrus porcine uterus. In addition, the distribution of β-adrenoceptors between muscle layers was characterized by means of adenylate cyclase activity assay, cyclic AMP assay and [3H]dihydroalprenolol binding studies. In the cornu, isoprenaline, adrenaline and noradrenaline inhibited the spontaneous contraction of longitudinal and circular muscles but longitudinal muscle was more sensitive to catecholamines than was circular muscle. The inhibitory response to isoprenaline was antagonized by propranolol (300 nM) or (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). The rank order of potency was isoprenaline≧adrenaline>noradrenaline. The β2-adrenoceptor-selective agonist, clenbuterol, was more potent than xamoterol (β1-selective) and (±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344; β3-selective) to inhibit the spontaneous contraction of longitudinal muscles. Isoprenaline increased adenylate cyclase activity in both muscle layers, but the activity in the longitudinal muscle was greater than that in the circular muscle. Cyclic AMP production by isoprenaline was also more conspicuous in the longitudinal muscle than in the circular muscle. Although both muscle layers contained a single class of [3H]dihydroalprenolol binding sites with similar Kd values (longitudinal muscle, 3.1±0.94 nM, n=4; circular muscle, 2.4±0.73 nM, n=4), Bmax in the longitudinal muscle (175.7±32.8 fmol/mg protein, n=4) was significantly higher than that in the circular muscle (53.1±5.1 fmol/mg protein, n=4). As previously reported [Br. J. Pharmacol. 130 (2000) 79], 5-HT also inhibited the spontaneous contraction of both muscle layers from the cornu and the 5-HT7 receptor antagonist, 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydro-benzo[cd]indol-2(1H)-one (DR4004; 100 nM, n=4) blocked the 5-HT-induced inhibition of spontaneous contractions in the circular muscles, and reversed the less marked inhibition in the longitudinal muscles. In other regions of the uterus (corpus and cervix), 5-HT inhibited the spontaneous contraction of the circular muscles but contracted the longitudinal muscle strips. On the other hand, isoprenaline caused muscle layer-dependent inhibition (longitudinal muscle>circular muscle) in both regions, and the responsiveness tended to increase toward the cervix. In conclusion, β2-adrenoceptors are present heterogeneously in the porcine uterus (longitudinal muscle>circular muscle) and share the inhibition of uterine contractility with 5-HT7 receptors in a layer-dependent manner (longitudinal muscle: β2-adrenoceptors, circular muscle: 5-HT7 receptors).

The role of myometrial β-adrenoreceptors (characterized by specific binding of (-)-[3H]-dihydroalprenolol) in the control of uterine relaxation and cAMP production was investigated by determining the concentrations of isoproterenol which produced half the maximal relaxation and half the maximal cAMP production, respectively. Under optimal but different conditions for each, half-maximal concentrations for both agonist-binding to β-adrenoreceptors and cAMP production by intact muscle strips were 20 times greater than half-maximal relaxation. Coupling between theβ -adrenoreceptor and adenylate cyclase was characterized in cell-free preparations where, as previously reported (Krall and Korenman, 1979), enzyme stimulation by the agonist had an absolute guanylyl nucleotide requirement. There was good agreement between the isoproterenol requirements for half-maximal hormone stimulation of adenylate cyclase and half-maximal (3-adreno-receptor binding of the agonist in the presence of guanylyl nucleotide. Thus, in myometrium,β -adrenoreceptors were directly coupled to cAMP production and tightly coupled to relaxation through a highly efficient mechanism. As a consequence, occupancy of less than 10% of the receptors and a commensurately small elevation in cAMP levels could account for one-half of the maximal relaxing effects of isoproterenol.

The β-adrenergic catecholamine agonist, isoproterenol, and the guanylyl nucleotide, GTP, had synergistic effects in elevating adenylate cyclase activity in uterine smooth muscle cell homogenates. The guanylyl nucleotide-dependent step included an increase in enzyme sensitivity to Mg2+. The Mg2+ requirements of the enzyme were independent of the ATP-substrate concentration, and the guanylyl nucleotidedependent activation step did not require either the divalent cation or enzymatic activity. These properties are most consistent with a two-stage enzyme model in which the equilibrium between Mg2+-sensitive and insensitive forms is modulated by guanylyl nucleotide. The activated enzyme state was transient and reversible, but reversion to the basal state probably required hydrolysis of GTP. Guanylyl nucleotide regulation extended to the interaction of isoproterenol with the smooth muscle cell β-adrenergic catecholamine receptor, but this effect was probably separate from the enzyme activation step.