The regulation of Phospholipase C (PLC)delta activity remains obscure. These studies show that PLCdelta1 activity is significantly enhanced by both guanosine thiotriphosphate (GTPgammaS) and Clostridium botulinum exoenzyme C3 (C3) but not by aluminium fluoride. C3 ADP ribosylated a 21-kDa protein in the PLCdelta1 preparation and Western blotting identified rhoA in these samples. RhoA acts as an inhibitory modulator of PLCdelta activity.

Rat models of cardiac hypertrophy are characterised by a shift in left ventricular myosin isoform from V1 (adult) to V3 (foetal), the latter being associated with a slowing of the acto-myosin ATPase rate. The aim of this study was to examine hypertrophy effects on relaxation by investigating a chemically skinned cardiac preparation from the SHR, where all the cellular membranes are rendered non-functional allowing the myofibrils to be studied in isolation. On comparison, following photolysis of the photolabile caged Ca2+ chelator diazo-2, it can be seen that the SHR fibres relax at a slower rate than their age-matched WKY counterparts. We suggest that, since the thin filament regulatory proteins seem not to be affected by cardiac hypertrophy in the rat, this result can be attributed to the shift in left ventricular myosin isoforms. The reduced relaxation rate in the SHR could be the result of a slowing of the dissociation of actin and myosin during the cross-bridge cycle. These results have previously been published in abstract form [1].

Cardiac hypertrophy in the guinea-pig is not accompanied by a large shift in the expression of the predominant isoform of myosin in the left ventricle; however, in this species, thin filament proteins do change. We examined the relaxation, following laser flash photolysis of the photolabile caged Ca2+ chelator diazo-2, of a skinned trabecular preparation from the left ventricle of guinea-pigs that had undergone abdominal aortic banding. Sham-operated animals were used as controls; no guinea-pigs showed any signs of heart failure. We report that mild cardiac hypertrophy does not affect the relaxation rate of Triton-skinned trabeculae from the guinea-pig. However, there was a 35% reduction in the maximum force generated by trabeculae from the left ventricle of the abdominal aortic-banded animals. Additionally, alterations in key troponin subunits occur in the left ventricle of guinea-pigs with mild hypertrophy. We conclude that the thin filament protein changes do not influence trabecular relaxation rates, even though they probably affect maximal force generation. The cellular membrane systems of the intact guinea-pig heart, which were not a factor in this present study, appear to have an important role in the altered cardiac relaxation rates seen in hypertrophy.

No abstract

Phosphatidylinositol4,5-bisphosphate is hydrolysed by phospholipase C (PLC) to form inositol 1,4,5-trisphosphate and diacylglycerol. Each of these products acts as a second messenger in a variety of cellular responses including contraction and cell proliferation [ 1,2]. The PLC family of enzymes consists of three isoforms (P,y,S) which are differentially regulated and expressed. PLCP has been demonstrated to be regulated by heterotrimeric G proteins of the Gq family or by Py subunits [3,4] whereas PLCy is tyrosine phosphorylated by growth factors [5]. The regulation of PLCG isoforms remains obscure although there is increasing evidence to suggest the involvement of GTP-binding proteins [6,71.Partially purified PLC& was isolated from bovine aortic cytosol by sequential elution through Q-sepharose and heparin agarose chromatography columns, using a linear sodium chloride gradient (50-55OmM) and identified immunologically by western blotting. PLC activity was measured using a method based on that described by Waldo et a1 [8] and using [3H]-phosphatidylinositol as a substrate. hmunoabsorption of PLC& from this sample resulted in a reduction in PLC activity of 89+3% (n=3) indicating that this sample is not contaminated with other isoforms of PLC, known or unknown.in the regulation of PLCGI activity samples were preincubated with G T W , a non-hydrolysable analogue of GTP, and PLC activity was measured. The resting PLC activity was consistently and significantly enhanced by the addition of GTPys ( Figure 1). but not by aluminium fluoride, suggesting the involvement of a monomeric GTP-binding protein.In order to determine whether GTP-binding proteins have a role ** I 300 T I T h control GTPVs Figure 1. Effect of GTPys on PLCGI activity. PLCGl samples were pre-incubated for 30 minutes at 37°C with 100lM G T W prior to measurement of PLC activity. Control activity was expressed as 100% and the GTPys stimulated activity was expressed as a % of the control. Data are mean f sem derived from six different aortae. ** p < 0.01To confirm the presence of a GTP-binding protein in the PLC& fraction, GTP-binding of column fractions were measured using [%]-GTPyS. These results demonstrated that the [35S]-GTpuS binding profile generated by the elution of the sample from heparin-agarose correlated exactly with that of PLCGI activity.Clostridium botulinum exoenzyme C3 (exoenzyme C3), which selectively ADP-ribosylates and inactivates rho GTPases also produced a significant increase in PLCGI activity. (Figure 2), *** I Control Exoenzyme C3 Figure 2. Effect of exoenzyme C3 on PLCGI activity. P E G , was preincubated for 1 hour with exoenzyme C3 (20pg/ml) and NAD prior to measuring PLC activity. Control activity is expressed as 100% and exoenzyme C3-stimulated activity is expressed as % of control. Data represents mean f sem from 4 separate aortae. *** p< 0.001.whereas incubation with NAD alone had no effect on PLC activity. Similarly exoenzyme C3 consistently ADP-ribosylated a 21kDa protein in the PLC& samples (n=3, data not shown) and wes...