CPA Concentration Research Articles

Intracellular cross‐talk between receptors coupled to phospholipase C via pertussis toxin‐sensitive and insensitive G‐proteins in DDT1MF‐2 cells

1. The effect on intracellular free calcium concentration ([Ca2+]i) of simultaneous activation of receptors coupled to phospholipase C via pertussis toxin (PTX)-sensitive and -insensitive G-proteins has been investigated in the hamster vas deferens smooth muscle cell line, DDT1MF-2. 2. In fura-2-loaded DDT1MF-2 cells, activation of adenosine A1-receptors (which are linked to PTX-sensitive G-proteins) with a maximal concentration of N6-cyclopentyladenosine (CPA; 300 nM) increased [Ca2+]i from 121 +/- 5 nM to 254 +/- 20 nM (n = 8). These experiments were performed in the presence of extracellular Ca2+. Stimulation of histamine H1-receptors (which are linked to PTX-insensitive G-proteins) with a low concentration of histamine (1 microM) increased [Ca2+]i from 128 +/- 8 nM to 150 +/- 13 nM (n = 8). When combined, CPA (300 nM) and histamine (1 microM) synergistically raised [Ca2+]i from 134 +/- 6 nM to 607 +/- 61 nM (n = 8). 3. Removal of extracellular Ca2+ (experiments performed in Ca(2+)-free buffer containing 0.1 mM EGTA) had no effect on the synergistic interaction between CPA (300 nM) and histamine (1 microM). 4. The addition of maximal concentrations of CPA (300 nM) and histamine (100 microM) resulted in a rise in [Ca2+]i which was additive when compared to the Ca2+ responses obtained with the two agonists alone. Low (30 nM) and subthreshold (3 nM) concentrations of CPA did not alter the Ca2+ response elicited by maximal concentrations of histamine (100 microM). 5. Subthreshold concentrations of CPA (3 nM) and low concentrations of histamine (1 microM) elicited synergistic rises in [Ca2+]i. 6 Synergistic Ca2+ responses were not observed between histamine Hl- and ATP-receptors when cells were simultaneously stimulated with either 1 microM or 10 microM of each agonist.7 These data suggest that adenosine A1-receptors linked to PTX-sensitive G-protein(s) and histamine H14-receptors linked to PTX-insensitive G-proteins interact synergistically to raise [Ca2+]i. In contrast,activation of ATP-receptors which are linked to PTX-insensitive G-protein(s) do not interact synergically with histamine H1-receptors.

Potassium-evoked efflux of transmitter amino acids and purines from rat cerebral cortex

Repeated applications of elevated K + (50 or 75 mM) in cerebral cortical cup superfusates was used to evoke an efflux of γ-aminobutyric acid (GABA), glutamate, aspartate, glycine, adenosine, and inosine from the in vivo rat cerebral cortex. K + (50 mM) significantly elevated GABA levels in cup superfusates but had little effect on the efflux of glutamate, aspartate, glycine, adenosine, or inosine. K + (75 mM) significantly enhanced the efflux of GABA, aspartate, adenosine, and inosine and caused nonsignificant increases in glutamate and glycine efflux. The adenosine A 1 receptor agonist N 6-cyclopentyladenosine (CPA), applied in cup superfusates at a concentration of 10 −10 M had no effect on either basal or K +-evoked release of any of the amino acids or purines measured. At 10 −6 M CPA significantly enhanced aspartate release, and depressed GABA efflux. The selective A 2 adenosine receptor agonist 2-p(2-carboxyethyl) phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS 21680) (10 −8 M) was without effect on either basal, or K +-evoked, efflux of amino acids or purines. The enhancement of aspartate (an excitotoxic amino acid) efflux by higher concentrations of CPA is likely due to activation of adenosine A 2b receptors. This observation may be of relevance when selecting adenosinergic agents to treat ischemic or traumatic brain injuries. Overall, the results suggest that effects of adenosine receptor agonists on K +-evoked efflux of transmitter amino acids from the in vivo rat cerebral cortex may not be comparable to those observed with in vitro preparations.

Excitatory transmitter amino acid release from the ischemic rat cerebral cortex: effects of adenosine receptor agonists and antagonists.

The effects of selective adenosine receptor agonists [N6-cyclopentyladenosine (CPA) and N-ethylcarboxamidoadenosine (NECA)] and antagonists [8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazoline-5-im ine (CGS-15943A)] on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four-vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug-treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10(-10) M) and NECA (10(-9) M) significantly inhibited the ischemia-evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia-evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10(-6) M) or NECA (10(-5) M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia-evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS-15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia-evoked release of excitatory neurotransmitter amino acids via high-affinity A1 receptors, whereas coactivation of lower-affinity A2 receptors may block (or reverse) the A1-mediated response.

Transient mass transfer processes during the perfusion of a biological organ with a cryophylactic agent solution

An analytical study was performed for the mass transfer processes which occurs during a typical CPA introduction protocol in a biological organ. In such a protocol the concentration of CPA in the perfusate changes linearly with time to a maximal value and is then kept at that value for an additional period of time. Numerical solutions to the Kedem-Katchalsky equations for mass transfer processes in an organ modeled by a large number of typical Krogh tissue units were found. The solutions indicate that several phenomena possibly harmful to the organ occur in the tissue as a function of H, the rate of increase in the CPA concentration.

Biochemical and immunological characterization of ceruloplasmin genetic variants

The results of biochemical and immunological characterization of five electrophorctic variants of human serum ceruloplasmin in Negroes are described. The variants were found to differ in the surface charge of the protein moeities of their molecules. No difference in their molecular weights could, however, be ascertained. The specific activities of the variants were strictly similar. Compared to the normal form CpB, CpA is more resistant and Cp Bpt is more susceptible to inhibition by either cyanide or azide of their oxidase activity while CpNH and CpA appear to resemble CpB in this regard. Citrate exerts uniform inhibition on all variants.Based on the differences in susceptibility to inhibition, the relative concentration of CpA to CpB protein in the CpAB heterozygote has been estimated to be 2:1. This may indicate that alleles at the ceruloplasmin locus (Cp) determine ceruloplasmin synthesis with different efficiencies. However, except for the Cp Bpt variant, the total level of ceruloplasmin protein in the serum appears to be regulated by a mechanism independent of the Cp phenotype and extrinsic to its structural genes.The objectives of these studies have been (A) to establish the distinctiveness of ceruloplasmin variants, (B) to provide means of identification and definition of new variants which may not differ electrophoretically from each other or from the variants already described, e.g. through differences in their inhibition profile, (C) to gather and assemble the necessary information to construct a model for control of quantitative expression of ceruloplasmin protein.