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We examined the effects of purines and the pyrimidine UTP on cellular proliferation in the human astrocytoma cell line 1321N1. Treatment of cultured cells with 100 microM ATP or 2-chloroadenosine (2-CA) resulted in significant reductions in cell numbers after 2 days, whereas adenosine (ADO) exhibited a slower time course of inhibition of cell growth. Treatment with 100 microM UTP had no effect on cell numbers. 2-Chloroadenosine but neither ATP nor ADO resulted in an increase in cell death rates. A significant portion of the inhibitory response to ATP, ADO, or 2-CA was sensitive to the purine nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioguanosine, suggesting that uptake into cells was required for the inhibitory response. At least the majority of the observed responses to purines was not mediated by P1 (adenosine) receptors, because effects of ATP, ADO, or 2-CA were not affected by treatment of cells with the P1 receptor antagonist 8-(p-sulfophenyl)-theophylline. The absence of any known P2 (nucleotide) receptors in 1321N1 cells, coupled with the failure of the relatively stable ATP analog adenosine 5'-O-(3-thiotriphosphate) to alter cell growth rates, suggests that ATP acts indirectly to inhibit proliferation via one or more metabolic products. Although intracellular effects of purine nucleosides should be taken into account in future studies using 1321N1 cells, our findings also suggest 1321N1 cells as an excellent model for intracellular actions of nucleosides.

The molecular and pharmacological characteristics of muscarinic receptor subtypes in the rat parotid acinar cell line, PAR-C5, were determined and compared with native rat parotid glands to evaluate the PAR-C5 cell line as a model to study receptor-mediated secretion. Reverse transcription-polymerase chain reaction (RT-PCR) identified mRNAs for M(3), M(4), and M(5) receptor subtypes in both PAR-C5 cells and parotid glands. Specific [N-methyl-(3)H]scopolamine binding in PAR-C5 and parotid membranes was to a single class of sites with mean K(D) values of 0.38 and 0.64 nM, respectively. Binding affinities (K(I) values) of muscarinic receptor subtype-selective drugs were obtained in side-by-side experiments comparing PAR-C5 cells with parotid glands. Nonlinear regression analysis indicated that competition binding curves for drugs in PAR-C5 cells and parotid glands fit best to a one-site binding model. K(I) values (nM) in PAR-C5 cells and parotid glands, respectively, for atropine (1.0, 2.1), darifenacin (1.2, 2.0), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (2.9, 2.4), tripitramine (220, 180), pirenzepine (320, 720), and methoctramine (1400, 1700) were consistent with their known affinities at the M(3) receptor subtype. Affinities (K(B) values) of muscarinic receptor subtype-selective drugs for blocking methacholine-stimulated Ca(2+) mobilization were determined to show which subtype mediates Ca(2+)-dependent secretion in Fura-2-loaded PAR-C5 cells. K(B) values (nM) for atropine (0.44), 4-DAMP (0.38), pirenzepine (140), and methoctramine (320) for blocking Ca(2+) responses correlated well with their known affinities at the M(3) receptor (r(2) = 0.99). These results show that at the level of mRNA, receptor protein and function, PAR-C5 cells and parotid glands are similar, establishing PAR-C5 cells as an important model for muscarinic receptor-mediated secretion.

We consider the problem of well-posedness and regularity of solutions for a dynamic von Kármán plate which is clamped along one portion of the boundary and which experiences boundary damping through “free edge” conditions on the remainder of the boundary. We prove the existence of unique strong solutions for this system