Nerve growth factor inhibits PC12 cell PDE 2 phosphodiesterase activity and increases PDE 2 binding to phosphoproteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66241/1/j.1471-4159.2001.00133.x.pd

Inhibition of PKC activity blocks the increase of ET(B )receptor expression in cerebral arteries

BACKGROUND: Previous studies have shown that there is a time-dependent upregulation of contractile endothelin B (ET(B)) receptors in middle cerebral arteries (MCA) after organ culture. This upregulation is dependent on mitogen-activated protein kinases and possibly protein kinase C (PKC). The aim of this study was to examine the effect of PKC inhibitors with different profiles on the upregulation of contractile ET(B )receptors in rat MCA. Artery segments were incubated for 24 hours at 37°C. To investigate involvement of PKC, inhibitors were added to the medium before incubation. The contractile endothelin-mediated responses were measured and real-time PCR was used to detect endothelin receptor mRNA levels. Furthermore, immunohistochemistry was used to demonstrate the ET(B )receptor protein distribution in the MCA and Western blot to measure which of the PKC subtypes that were affected by the inhibitors. RESULTS: The PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and PKC inhibitor 20–28 attenuated the ET(B )receptor mediated contractions. Furthermore, Ro-32-0432 and bisindolylmaleimide I decreased ET(B )receptor mRNA levels while PKC inhibitor 20–28 reduced the amount of receptor protein on smooth muscle cells. PKC inhibitor 20–28 also decreased the protein levels of the five PKC subtypes studied (α, βI, γ, δ and ε). CONCLUSION: The results show that PKC inhibitors are able to decrease the ET(B )receptor contraction and expression in MCA smooth muscle cells following organ culture. The PKC inhibitor 20–28 affects the protein levels, while Ro-32-0432 and bisindolylmaleimide I affect the mRNA levels, suggesting differences in activity profile. Since ET(B )receptor upregulation is seen in cerebral ischemia, the results of the present study provide a way to interfere with the vascular involvement in cerebral ischemia

Oxidant Sensing by Protein Kinases A and G Enables Integration of Cell Redox State with Phosphoregulation

The control of vascular smooth muscle contractility enables regulation of blood pressure, which is paramount in physiological adaptation to environmental challenges. Maintenance of stable blood pressure is crucial for health as deregulation (caused by high or low blood pressure) leads to disease progression. Vasotone is principally controlled by the cyclic nucleotide dependent protein kinases A and G, which regulate intracellular calcium and contractile protein calcium sensitivity. The classical pathways for activation of these two kinases are well established and involve the formation and activation by specific cyclic nucleotide second messengers. Recently we reported that both PKA and PKG can be regulated independently of their respective cyclic nucleotides via a mechanism whereby the kinases sense cellular oxidant production using redox active thiols. This novel redox regulation of these kinases is potentially of physiological importance, and may synergise with the classical regulatory mechanisms

Alteration of vascular reactivity in heart failure: role of phosphodiesterases 3 and 4

BACKGROUND AND PURPOSE: This study examined the role of the main vascular cAMP-hydrolysing phosphodiesterases (cAMP-PDE) in the regulation of basal vascular tone and relaxation of rat aorta mediated by β-adrenoceptors, following heart failure (HF). EXPERIMENTAL APPROACH: Twenty-two weeks after proximal aortic stenosis, to induce HF, or SHAM surgery in rats, we evaluated the expression, activity and function of cAMP-PDE in the descending thoracic aorta. KEY RESULTS: HF rat aortas exhibited signs of endothelial dysfunction, with alterations of the NO pathway, and alteration of PDE3 and PDE4 subtype expression, without changing total aortic cAMP-hydrolytic activity and PDE1, PDE3 and PDE4 activities. Vascular reactivity experiments using PDE inhibitors showed that PDE3 and PDE4 controlled the level of PGF(2α)-stimulated contraction in SHAM aorta. PDE3 function was partially inhibited by endothelial NO, whereas PDE4 function required a functional endothelium and was under the negative control of PDE3. In HF, PDE3 function was preserved, but its regulation by endothelial NO was altered. PDE4 function was abolished and restored by PDE3 inhibition. In PGF(2α)-precontracted arteries, β-adrenoceptor stimulation-induced relaxation in SHAM aorta, which was abolished in the absence of functional endothelium, as well as in HF aortas, but restored after PDE3 inhibition in all unresponsive arteries. CONCLUSIONS AND IMPLICATIONS: Our study underlines the key role of the endothelium in controlling the contribution of smooth muscle PDE to contractile function. In HF, endothelial dysfunction had a major effect on PDE3 function and PDE3 inhibition restored a functional relaxation to β-adrenoceptor stimulation

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice

International audienceADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs

Megakaryocytes form linear podosomes devoid of digestive properties to remodel medullar matrix

International audienceBone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them