The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase C␣, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V 4؉ -OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V 4؉ -OOH, PLD1 was tyrosinephosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.

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These results indicate that the enhanced expression of Gi alpha-2 and Gi alpha-3 precedes the development of blood pressure in DOCA-salt-induced hypertension. It can thus be suggested that the increased levels of Gi proteins and resulting decreased levels of cAMP may be one of the factors that contribute to the impaired cardiac contractility and increased vascular tone in DOCA-salt hypertension.

We have previously demonstrated a decreased expression of Gi alpha 2 protein in platelets from spontaneously hypertensive rats that was associated with an altered responsiveness of adenylyl cyclase to hormone stimulation and inhibition. In the present studies, we have used platelets from hypertensive patients and examined the hormonal regulation of adenylyl cyclase as well as the levels of G proteins and their modulation by antihypertensive drug therapy. We performed these studies in platelets from four groups of subjects: normotensive subjects (group 1), untreated mildly essential hypertensive patients (group 2), and treated moderately to severely hypertensive patients whose blood pressure was uncontrolled (group 3) or controlled with drug treatment (group 4). GTP gamma S, 5'-(N-ethylcarboxamido)adenosine (NECA), and prostaglandin E1 stimulated adenylyl cyclase activity to a greater extent in hypertensive patients (group 2). This effect was partially corrected (by approximately 50% to 80%) in the patients under antihypertensive drug therapy (groups 3 and 4). In addition, inhibition of adenylyl cyclase mediated by a ring-deleted analogue of atrial natriuretic factor (C-ANF4.23) observed in control normotensive subjects was blunted in hypertensive patients (group 2) and was not corrected in treated patients. Gi alpha levels determined by immunoblotting were in the same range for the four groups, whereas Gi alpha 2 and Gi alpha 3 levels were decreased by 70% and 60%, respectively, in hypertensive patients (group 2) compared with normotensive subjects. Antihypertensive drug therapy (groups 3 and 4) partially restored Gi alpha 2 levels toward normal (group 1) by about 60% and 70%, respectively; however, the reduced Gi alpha 3 levels in group 2 hypertensive patients were not improved in group 3 but were raised toward normal levels in group 4 by about 55%. These results suggest that the altered responsiveness of platelet adenylyl cyclase to hormones in hypertension and the normalization of the response with antihypertensive drug therapy could partly be due to the ability of the latter to modulate Gi alpha protein expression. These effects on platelet function may underlie the beneficial effects of antihypertensive agents on some of the complications of hypertension.