Characterization of extracellular nucleotide-induced Mac-1 (alphaM beta2 integrin) surface expression on peripheral blood leukocytes.

Extracellular nucleotides, released during vascular injury, stimulate hematopoietic cells resulting in various physiological responses. We have determined that nucleotides can stimulate the expression of Mac-1 on peripheral blood leukocytes. ATP stimulated the expression of Mac-1 in a time- and dose-dependent manner with maximum expression occurring in 5 min at 10 microM ATP. This increase in surface expression was observed in monocytes and granulocytes was dose-dependent and was comparable in extent to the increase induced by the chemotactic peptide, formyl-Met-Leu-Phe. Other nucleotides including 2-MeSADP, ADP, UTP, and 2MeSATP had similar effect. Nucleotide-mediated stimulation of Mac-1 expression in granulocytes was completely inhibited by Ro-31-8220, a specific inhibitor of protein kinase C, while variable inhibition was observed in monocytes. These results demonstrate the stimulation of peripheral blood leukocytes by nucleotides causing an increased surface expression of Mac-1 which may be mediated by the activation of protein kinase C.

Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells.

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 +/- 6 nM), much less for UTP and ADP (approximately 1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.

Characterization of P2 purinergic receptors on human erythro leukemia cells.

We have investigated the nature of the nucleotide receptors on human erythro leukemia (HEL) cells, a cell line with some megakaryocytic properties, using a combination of pharmacological, photoaffinity labeling, and molecular biological techniques. Fura-2 loaded HEL cells responded to 2-methylthio ATP, ATP, 2-methylthio ADP, ADP and UTP with an increase in intracellular calcium. 2 Methylthio ADP was the most potent agonist. When external calcium was chelated with EDTA, calcium responses were observed indicating the mobilization of intracellular stores. These responses showed evidence of both homologous and heterologous receptor desensitization. In photoaffinity labeling experiments, beta-[32P]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to M(r) approximately 55 kDa (doublet) and approximately 43 kDa. Labeling of approximately 55 kDa proteins was specifically inhibited by ADP, while that of the approximately 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA library with mouse P2U cDNA revealed that the P2U receptor from HEL cells is identical to the previously cloned human P2U receptor. These experiments suggest that the HEL cells contain a P2Y purinoceptor responding to ADP, in addition to a P2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.

A receptor that subserves reovirus binding can inhibit lymphocyte proliferation triggered by mitogenic signals.

A novel surface receptor complex involved in inhibition of T-cell proliferation is described. Biochemical isolation revealed two non-covalently associated proteins of about M(r) 65,000 (p65) and 95,000 (p95). These polypeptides may be related. The p65 form is expressed after cellular activation and replication and is recognized by monoclonal antibody (mAb) 87.92.6 or reovirus hemagglutinin as unnatural ligands. The p95 species is associated with tyrosine kinase enzymatic activity. Receptor ligation results in rapid alteration of the phosphotyrosine content of cellular substrates, and this activity correlates with antiproliferative effects. The inhibition of proliferation is a time-dependent reversible arrest at the G1-S phase of the cell cycle. Activation through the T-cell receptor, protein kinase C, or addition of cytokines does not reverse the antiproliferative effect. This receptor complex may define novel features of T-cell proliferation.

Brahmighritham, an Ayurvedic herbal formula for the control of epilepsy.

Brahmighritham is an Ayurvedic herbal recommended for the control of epilepsy. The preparation of the drug is described. It was tested for its oral effectiveness in controlling pentylenetetrazole-induced seizures in male albino rats and was compared with benzdiazepam. Alterations in the EEG pattern and gross neurological function were measured or rated 60 min after pentylenetetrazole administration. Thirty-day pretreatment with both Brahmighritham and benzdiazepam served to make the rats more insensitive to epileptogenic events.