guanylyl-imidodiphosphate and cis-vaccenic-acid

beta-Adrenoceptor (beta AR) responsiveness, receptor density, receptor-G protein coupling, and the possible role of membrane fluidity in receptor-G protein coupling were investigated in the rat aorta with age. The beta AR agonist isoproterenol (ISO) produced relaxation of KCl-induced aortic contractions by 97%, 21%, and 0% in aortae from 1- 6-, and 24-month-old Fischer 344 rats, respectively. Forskolin completely relaxed the contractions at all ages. beta AR density was determined in aortic membranes by saturation binding of 125I-cyanopindolol (125I-CYP). beta AR density was 76, 52, and 47 fmol/mg of protein in 1-, 6-, and 24-month-old rats, respectively. To investigate beta AR coupling to G proteins, displacement by ISO of 125I-CYP binding was determined in aortic membranes in the presence and absence of the GTP analog guanosine-5'-(beta gamma-imido)triphosphate [Gpp(NH)p] (0.1 mM). The effect of Gpp(NH)p on the ISO displacement curve for 125I-CYP binding was greatest in 1-month-old rats and decreased markedly with age. In 1-month-old aorta, in the absence of Gpp(NH)p the ISO displacement curve was biphasic and two affinity constants were determined (KH - 0.061 microM and KL = 2.4 microM). In the presence of Gpp(NH)p the ISO displacement curve was monophasic (Kd - 0.72 microM). In 6-month-old aorta, whereas an effect of Gpp(NH)p on the ISO displacement curve could still be observed [in the absence of Gpp(NH)p, KH = 0.2 microM and KL = 3.5 microM; in the presence of Gpp(NH)p, Kd - 0.83 microM], the affinity constant for high affinity agonist binding and the percentage of receptors with high affinity for agonist were decreased significantly. In 24-month-old aorta there was no effect of Gpp(NH)p on the ISO displacement curve and a single affinity constant was detected [0.7 microM and 0.8 microM in the presence and absence of Gpp(NH)p, respectively]. The presence of two affinity constants for ISO in 1- and 6-month-old aorta in the absence of Gpp(NH)p and single affinity constants in the presence of Gpp(NH)p presumably represent the G protein-coupled and uncoupled states of the beta ARs, which are not observed in 24-month-old aorta. The ability of the beta AR to form the high affinity nucleotide-sensitive complex with the agonist was restored by treatment of the membranes with cis-vaccenic acid, which increases the fluidity of the membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Aging; Animals; Aorta; GTP-Binding Proteins; Guanylyl Imidodiphosphate; In Vitro Techniques; Iodocyanopindolol; Male; Oleic Acids; Pindolol; Precipitin Tests; Rats; Rats, Inbred F344; Receptors, Adrenergic, beta; Vasodilation

Guanine nucleotides couple receptors to stimulate or inhibit adenylate cyclase as well as regulate binding of neurotransmitters. To explore the relationship between these different functions of guanosine 5'-triphosphate (GTP), rat brain membranes were preincubated in 50 mM sodium acetate, pH 4.5, which increased GTP regulation of 3H-opiate agonist binding. Assay of adenylate cyclase in the low pH-pretreated membranes revealed no loss of basal activity but a dramatic loss in fluoride- and guanylyl-5'-imidodiphosphate-stimulated activity, thus suggesting a loss in stimulatory guanine nucleotide coupling function. Manganese stimulation, which presumably occurs on the catalytic subunit of adenylate cyclase directly, was not affected by low pH treatment. In striatum, dopamine-stimulated adenylate cyclase was eliminated, but inhibition of adenylate cyclase by D-Ala2-Met5-enkephalinamide (D-Ala enk) was increased by low pH treatment. The effect of low pH on sodium fluoride-stimulated and D-Ala enk-inhibited adenylate cyclase could be reversed by addition of either cis-vaccenic acid or phosphatidylcholine to treated membranes, but the effect on GTP regulation of binding was not reversed by lipid incorporation. These results suggest that fundamental differences exist between membrane components which couple receptors to adenylate cyclase and those that regulate neurotransmitter binding.

Topics: Adenylyl Cyclases; Animals; Brain; Cell Membrane; Corpus Striatum; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hydrogen-Ion Concentration; Male; Membrane Fluidity; Membrane Lipids; Oleic Acids; Phosphatidylcholines; Rats; Rats, Inbred Strains; Receptors, Opioid; Sodium Fluoride; Viscosity