We have described a thyroid hormone receptor in synaptosomes of the chick embryo brain. To understand how the hormones exert their actions at this level, we performed a series of studies to demonstrate that this receptor could be linked to G proteins. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)(100 muM) lowered the binding capacity of the receptor high affinity site from 8.9 +/- 1.3 to 3.4 +/- 1.3 ng T3/mg protein, a finding consistent with the coupling of receptor to G proteins. Furthermore, ADP ribosylation with pertussis toxin showed that thyroid hormones induced a dose-dependent increase in the inactive alpha 0-subunit of the G0 protein. This effect was detected at 10 pM, with a maximal increase (mean +/- SEM, 50 +/- 3.6%) at 100 nM, and T4 was as effective as T3. Both hormones also decreased the intrinsic guanine triphosphatase activity of G proteins by lowering the binding of GTP to the alpha-subunit and their rate of hydrolysis. This inhibition was greater with T4 (25 +/- 5%) than with T3 (14 +/- 2%), suggesting that the former could be the more active hormone at the synaptosomal level. The effect on guanine triphosphatase activity confirms that the synaptosomal thyroid hormone receptor is coupled to a G(zero) protein. These results demonstrate that thyroid hormones increase or favor the ADP ribosylation of G alpha(zero) by pertussis toxin. Thus, they enhance the alpha(zero)-GDP form of the G(zero) protein, namely its inactive conformation. By decreasing the activity of this protein, these hormones may modulate the formation of second messengers in synaptosomes and intervene in the regulation of neuronal proliferation and differentiation induced by several factors. Therefore, thyroid hormones may exert their action on brain maturation at least in part by modulating G alpha(zero) through their synaptosomal receptor.
In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K+), and the presence of short and long lasting subconducting states. Its open probability Po increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca2+ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K+ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca2+] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca2+, Ba2+ or Mg(2+)-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by microM Ca2+ while its Ca2+ sensitivity was potentiated by millimolar ATP. However Mg2+ and calmodulin modulated its gating behavior. Ca2+ releasing drugs such as caffeine and ryanodine increased its Po. All these features are characteristics of the SR Ca2+ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
The involvement of various phosphodiesterases (PDEs) in controlling the time-dependent mechanical properties of guinea pig trachealis smooth muscles was determined by using different classes of PDE inhibitors as pharmacological tools. These drugs produced low amplitude and long-lasting dose-dependent relaxations on the resting tone with the following EC50 values: rolipram, 3 nM; indolidan, 0.11 microM; and zaprinast, 0.5 nM and 1 microM. These PDE inhibitors were 50% less active than 1 microM norepinephrine. The effects of the drugs were also tested on carbachol-induced contractions and norepinephrine-evoked relaxations. Zaprinast, but not rolipram nor indolidan, decreased the rate of rise of contraction, thus prolonging the time to reach the plateau by 75% without modifying the magnitude of the responses. Zaprinast and rolipram significantly increased the total length of the norepinephrine effect by 25 and 35%, respectively. Similar results were obtained in a dose-dependent manner on isoproterenol-induced relaxations. In contrast, a higher concentration of indolidan was required to affect the amplitude, duration, and time to peak of isoproterenol- or norepinephrine-induced relaxations. These results indicate that PDE IV (rolipram sensitive) and PDE I, and less likely PDE V (both zaprinast sensitive), are involved in the control of guinea pig airway contractile kinetics, whereas PDE III (indolidan sensitive) is essentially involved in the modulation of the resting tone. Four cytosolic isozymes were identified in bovine airway smooth muscles (ASMs); PDE I (calmodulin-dependent PDE), PDE II (cGMP-stimulated PDE), PDE IV (cAMP-specific and rolipram-sensitive PDE), and PDE V (cGMP-specific and zaprinast-sensitive PDE). Characterization of PDE isoforms present in the microsomal fraction by HPLC showed the presence of PDE IV, PDE V, and to a lesser extent PDE III. However, PDE III was not detected in ASM cytosol. Using newly synthesized radioligands, binding studies confirmed the low level of expression of PDE III and the presence of PDE IV. We conclude that PDE I controls the rate of contraction, whereas PDE V and PDE IV prolong the time of relaxation induced by NE. PDE V would control the ASM responsiveness by regulating the intracellular cGMP concentration, which in turn would both activate PKG and stimulate PDE II (cGS-PDE). Since the various isozymes of PDE are differently involved in the kinetic control of the mechanical events in ASM, they represent physiologically relevant and important pharmacological targets.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.