1 We have investigated the actions of NS1619, a putative activator of large conductance calciumactivated potassium channels (BKCa) by use of the patch-clamp technique on smooth muscle cells enzymatically isolated from the rat basilar artery.2 Using whole cell current-clamp to measure membrane potential, addition of 30 gM NS1619 produced cellular hyperpolarization, moving the membrane potential towards the calculated equilibrium potential for potassium. This hyperpolarization was rapidly reversed by IbTX (100 nM), a selective inhibitor of BKCa. 3 In whole cell recordings made from cells voltage-clamped at 0 mV using the perforated-patch technique, addition of NS1619 (10-30 gM) activated an outward current, which reversed following washout of NS1619. 4 This outward current was unaffected by application of either glibenclamide (5 gM), an inhibitor of ATP-sensitive potassium channels, or apamin (100 nM), an inhibitor of small-conductance calciumactivated potassium channels. However, this current was almost completely abolished by iberiotoxin (IbTX; 50-lOOnM). 5 Depolarizing voltage steps activated small outward currents from cells held at -15 mV. Application of NS1619 (10-30 gM) increased the size of these currents, producing a shift to the left of the currentvoltage (I-V) relationship. These currents were largely inhibited by IbTX (100 nM). 6 Measurements of the unitary amplitude of the single channels activated by NS1619 which could be resolved in whole cell recordings yielded a value of 5.6+0.14 pA at 0 mV. 7 NS1619 (10-30 gM) directly activated single channels contained in excised inside-out and outside-out membrane patches. In both configurations NS1619 (10-30 gM) rapidly increased the open probability of a large conductance calcium-dependent channel. The activation produced by NS1619 was calciumdependent and inhibited by external IbTX (100 nM). The unitary current amplitude was unaffected by NS1619. 8 By use of conventional whole cell recording methods and conditions that suppressed BKCa openings, outward potassium currents were activated by depolarizing potentials positive to -35 mV from a holding potential of -65 mV. NS1619 (10-30 gM) inhibited this current in a concentration-dependent manner. This inhibition was reversed following washout of NS1619, recovering to 60-90% of control values within 2 min. 9 Ba2+ currents, measured by conventional whole cell recording, were activated by depolarizing voltage steps from negative holding potentials. NS1619 (1-30 gM) inhibited the evoked current in a concentration-dependent manner, yielding an IC50 value of 7 gM with a Hill coefficient approaching unity. This inhibition was reversible, with the currents recovering to 65-100% of control values after washout of NS1619 for 2 min. 10 NS1619 (0.3-100 pM) induced concentration-dependent relaxation of basilar artery segments contracted with histamine/5-HT (IC50 = 12.5 + 2.0 gM; n = 4). This relaxation curve was shifted to the right, but not abolished, when the tissue was treated with a blocker of BKCa channels (IbTX; l...

We have investigated the potential of genistein, an estrogenic component of soy, when administered neonatally, to manifest a protective effect against chemically induced mammary cancer. Female Sprague-Dawley rats were treated on Day 2, 4, and 6 postpartum with genistein or dimethylsulfoxide (vehicle). To induce mammary carcinogenesis, all animals were subsequently exposed on Day 50 postpartum to dimethylbenz(a)anthracene. Animals treated neonatally with genistein had increased latency and reduced incidence and multiplicity of mammary tumors compared with vehicle-treated animals. Cell differentiation studies in mammary whole mounts revealed that neonatal genistein treatment resulted in decreased numbers of terminal end buds and increased numbers of lobular structures. A precocious maturation of undifferentiated terminal end buds to more differentiated lobules may account for neonatal genistein treatment protecting against chemically induced mammary cancer.

The endogenous cannabinoid, anandamide, has been suggested as an endothelium-derived hyperpolarizing factor (EDHF). We found that anandamide-evoked relaxation in isolated segments of rat mesenteric artery was associated with smooth muscle hyperpolarization. However, although anandamide-evoked relaxation was inhibited by either charybdotoxin (ChTX) or iberiotoxin, inhibition of the relaxation to EDHF required a combination of ChTX and apamin. The relaxations induced by either anandamide or EDHF were not inhibited by the cannabinoid receptor (CB 1 ) antagonist SRI41716A, or mimicked by selective CB 1 agonists. Thus, anandamide appears to cause smooth muscle relaxation via a CB 1 receptorindependent mechanism and cannabinoid receptor activation apparently does not contribute to EDHFmediated relaxation in this resistance artery.

1 The aim of the current study was to characterize which cannabinoid receptors, if any, are present on rat carotid artery smooth muscle. Additionally, the eects of cannabinoids on carotid artery tone, on cyclic AMP accumulation and on forskolin-induced relaxation were examined in the same tissue. 2 Stimulation of carotid arteries with forskolin (10 mM) signi®cantly increased cyclic AMP accumulation, an eect that was inhibited in a concentration-dependent manner by the cannabinoid receptor agonist, methanandamide. 3 Similar inhibition was seen with the CB 1 agonist HU-210 but this inhibition was not mimicked by the CB 2 agonist, WIN 55,2212-2. 4 The inhibitory eect of methanandamide on cyclic AMP accumulation was prevented by incubation of the arteries with pertussis toxin and was signi®cantly reduced by LY320135, a selective CB 1 antagonist, but not by SR 144528, a CB 2 -selective antagonist. 5 Methanandamide failed to relax carotid arteries pre-contracted with phenylephrine, but inhibited forskolin-induced relaxation of these arteries. This functional inhibition of relaxation by methanandamide was inhibited by CB 1 -selective (LY320135 and SR 141716A), but not a CB 2 -selective antagonist (SR 144528). 6 These data demonstrate the presence of functional G protein-linked cannabinoid receptors of the CB 1 subtype in the rat carotid artery, but show that these receptors inhibit cyclic AMP accumulation rather than cause relaxation.