Little is known about the relative stoichiometry of guanine nucleotide-binding (G) proteins relative to the effector systems to which they link. We addressed this question for the stimulatory G protein (Gs) linked to adenylate cyclase. Forskolin stimulates the catalytic subunit of adenylate cyclase (C), but it has a higher efficacy and potency when C also interacts with the G protein Gs. Accordingly, we measured high-affinity [3H]forskolin binding to intact cells to assay alpha s-C complexes. No high-affinity specific binding occurred with unstimulated cells. The beta-adrenergic agonist isoproterenol promoted the binding of [3H]forskolin to about 3000 sites per cell, suggesting that each receptor on average activates at least several Gs molecules. Activating Gs directly with cholera toxin maximally promoted [3H]forskolin binding to a similar number of sites, suggesting that this is the maximal number of alpha s-C complexes formed per cell. We conclude that each cell likely contains only a few thousand functional copies of C, and that the availability of C (rather than Gs, which exists in more than 100,000 copies per cell) is likely to be limiting for agonist stimulation of adenylate cyclase activity.
Amrinone, a new bipyridine derivative, exerts a positive inotropic action in experimental preparations and is effective when administered orally to dogs. To assess its immediate effects in man, we studied by cardiac catheterization the hemodynamic responses to amrinone (1.85 to 3.5 mg per kilogram given intravenously) in eight patients with congestive heart failure already receiving full doses of digitalis. the following statistically significant (P less than 0.01) effects were noted: cardiac index increased from a mean +/- 1 S.D. of 1.8 +/- 0.3 to 2.6 +/- 0.3 liters per minute per square meter; peak rate of left ventricular pressure rise rose from 849 +/- 233 to 1206 +/- 456 mm Hg per second; left ventricular end-diastolic pressure fell from 25 +/- 9 to 14 +/- 7 mm Hg; pulmonary-capillary pressure fell from 28 +/- 8 to 15 +/- 4 mm Hg; and right atrial pressure fell from 12 +/- 6 to 7 +/- 5 mm Hg. Mean heart rate was unchanged, and aortic mean pressure declined slightly (86 +/- 10 to 80 +/- 7 mm Hg, P less than 0.025). No toxicity was observed. Amrinone, whose mechanism of action has not yet ben defined, warrants further study as a possible treatment for heart failure.
In many eukaryotic cell types, receptor activation leads to the formation of inositol 1,4,5-trisphosphate (IP3) which causes calcium ions (Ca) to be released from internal stores. Ca release was observed in response to the muscarinic agonist carbachol by fura-2 imaging of N1E-115 neuroblastoma cells. Ca release followed receptor activation after a latency of 0.4 to 20 s. Latency was not caused by Ca feedback on IP3 receptors, but rather by IP3 accumulation to a threshold for release. The dependence of latency on carbachol dose was fitted to a model in which IP3 synthesis and degradation compete, resulting in gradual accumulation to a threshold level at which Ca release becomes regenerative. This analysis gave degradation rate constants of IP3 in single cells ranging from 0 to 0.284 s -I (0.058_ 0.067 s -1 SD, 53 cells) and a mean IP3 lifetime of 9.2 +-2.2 s. IPs degradation was also measured directly with biochemical methods. This gave a half life of 9 • 2 s. The rate of IP3 degradation sets the time frame over which IP3 accumulations are integrated as input signals. IP~ levels are also filtered over time, and on average, large-amplitude oscillations in IP3 in these cells cannot occur with period < 10 s.
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