Effect of hawthorn (Crataegus oxycantha) crude extract and chromatographic fractions on multiple activities in a cultured cardiomyocyte assay.

Extracts of hawthorn (Crataegus oxycantha) have become popular herbal supplements for their well-recognized cardiotonic effects. Many commercial preparations have been used successfully in the treatment of congestive heart failure, although the active principles within these extracts have yet to be conclusively identified. Several hawthorn preparations were studied and found to have negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay using unpaced cells. As compared to conventional cardiac drugs (i.e., epinephrine, milrinone, ouabain, or propranolol), hawthorn extract has a unique activity profile. Hawthorn extract appears to be anti-arrhythmic and capable of inducing rhythmicity in quiescent cardiomyocytes. Hawthorn extract does not cause beta-adrenergic receptor blockade at concentrations which cause negative chronotropic effects. Commercial hawthorn preparations, extracts prepared from dried leaves and those made from dried berries have similar chronotropic activities. When crude extracts are separated using size-exclusion chromatography, several fractions retain multiple cardiac activities. Assays with chromatographic fractions reveal that multiple dissimilar cardioactive components may exist within the extract, making the identification of individual active constituents more challenging.

Expression of a cloned P2Y purinergic receptor that couples to phospholipase C.

P2Y purinergic receptors previously have been shown to couple either to activation of phospholipase C through a pertussis toxin-insensitive mechanism or to inhibition of adenylyl cyclase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmacological data strongly suggest that multiple P2Y purinergic receptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] cloned a cDNA that, when expressed in frog oocytes, displayed the general pharmacological characteristics of a P2Y purinergic receptor but whose second messenger linkage was not resolved. We have now cloned the meleagrid (turkey) homologue of the previously cloned chick P2Y purinergic receptor and have stably expressed it in a heterologous human cell line (1321N1 astrocytoma cells) to establish its signaling properties. The purinergic receptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked activation of phospholipase C in 1321N1 cells stably expressing the meleagrid receptor. The order of potency of a series of analogues of ATP and ADP for stimulation of phospholipase C by the receptor expressed in 1321N1 cells [2MeSATP = 2-methylthio-ADP > adenosine 5'-O-(2-thio)diphosphate > ADP > 2-chloro-ATP = adenosine 5'-O-(3-thio)triphosphate > or = ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P2Y purinergic receptors in turkey erythrocytes and many other tissues and was markedly different from those of the P2U and P2X purinergic receptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergic agonists was not affected by preincubation of cells with pertussis toxin. In contrast to its marked effects on phospholipase C activity, 2MeSATP caused only a small and variable inhibition of cAMP accumulation. Ribonuclease protection analysis of turkey tissues showed that this P2Y purinergic receptor is most highly expressed in blood and brain. Taken together, these results indicate that a phospholipase-C-activating P2Y purinergic receptor has been cloned and stably expressed in 1321N1 astrocytoma cells.