SHORT COMMUNICATIONS Comparison of inositol phosphates formation and gene expression of Gq alpha subunit in cultured aortic smooth muscle cells from spontaneously hypertensive and Wistar Kyoto rats

Abstract Objecitve To explore whether phosphoinositide specific phospholipase C (PLC) activation via G protein in vascular smooth muscle cells (VSMCs) is altered in spontaneously hypertensive rats (SHR). Methods The VSMCs derived from aortae of SHR and Wistar Kyoto (WKY) rats were loaded for 48 hours with myo inositol. Inositol phosphate release was initiated by the addition of 10 5 mol/L norepinephrine in intact cells or by guanosine 5' 0 (3 thio tri sphosphate) (GTP gamma S) in permeabilized cells. In the meantime, growth arrested VSMCs were stimulated by 10% calf serum for 0, 30, 45, or 60 min, then gene expressions of Gq alpha subunit (G alph a q) were observed. Results There were no significant differences in inositol 1, 4,5 triphosphate (IP 3) level and expression of G alpha q mRNA between quiescent VSMCs from SHR and that from WKY. When stimulated by norepinephrine, IP 3 production increased transiently with a peak level at 10 s in VSMCs from WKY, and a rapid biphasic IP 3 response, which was significantly higher than that of WKY, in VSMCs from SHR had been observed. G proteins activated by GTP gamma S significantly raised IP 3 production in VSMCs from SHR compared to WKY (SHR vs WKY: 234.8%±29.2% vs 142.4%±12.0% of basal IP 3, P<0.05). In addition, the serum effect showed an significant increase in expression of G alpha q mRNA in VSMCs from SHR. Conclusions The hereditary factors are not the only variable regulating IP 3 metabolism and G alpha q gene expression. Influences of multi environmental factors such as vasoactive compounds, together with genetic predisposition, palys an important role in the highly sensitive response of IP 3 production and G alpha q gene over expression in SHR.

A SPX domain vacuolar transporter links phosphate sensing to homeostasis in Arabidopsis

Excess phosphate(Pi)is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells.We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level.We tested this hypothesis using Vacuolar Phosphate Transporter 1(VPT1),a SPX domain-containing vacuolar Pi transporter,as a model.Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules(InsPs)produced at high cellular Pi status.We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1.We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity.Taking together the results from detailed structure-function analyses,our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low,whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole.This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells.

ITPK1 is an InsP_(6)/ADP phosphotransferase that controls phosphate signaling in Arabidopsis.

In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. Here, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by Pi and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to Pi than lower inositol phosphates, a response conserved across kingdoms. With CE-ESI-MS we could separate different InsP7 isomers in Arabidopsis and rice, and identify 4/6-InsP7 and a PP-InsP4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP7, 5-InsP7 and InsP8 increase severalfold in shoots after Pi resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP6 compartmentalization. Notably, ITPK1 is critical for Pi-dependent 5-InsP7 and InsP8 synthesis in planta and its activity regulates Pi starvation responses in a PHR-dependent manner. Furthermore, we demonstrate that ITPK1-mediated conversion of InsP6 to 5-InsP7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP7 under low ATP. Collectively, our study provides deeper insights into Pi-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.