Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.

No abstract

This study investigates the development of sexually dimorphic sensitivity of the GH system to α₂-adrenergic stimulation and GH feedback in the rat. Sensitivity to α₂-adrenergic stimulation was tested with clonidine (CLN, an α₂-adrenergic agonist) which stimulates GH release in the adult male rat. Feedback was examined by testing whether human (h)GH suppressed the CLN-induced GH surge as previously demonstrated in adult male rats. The integrity of the pituitary and its capacity to respond to stimulation was tested at the end of the experiment by perifusing with GRF. Studies were conducted using a hypothalamic-pituitary coperifusion system which allows incubation of these tissues without the confounding influences of peripheral hormonal and extra-hypothalamic neural factors. Tissue from prepubertal rats of 10, 20, 25 and 30 days of age, 50-day-old and adult rats (90-100 days) were evaluated. Results indicate that tissue from both male and female rats is sensitive to α₂-adrenergic stimulation at 10 days of age. In male tissue, there is an increase in GH release in response to CLN until 30 days of age, after which a slight decline in sensitivity occurs by 50 days of age and is maintained in adulthood. In regard to GH release from female tissue, a GH surge occurs in response to CLN until 30 days of age. At 50 days and in adulthood, this response is substantially diminished. Additionally, there is a profound sexual dimorphism in the capacity of hGH to suppress the CLN induced GH surge. In tissue from male rats, by 20 days of age there is an apparent GH-associated inhibition of the CLN-induced GH surge which is significant by 25 days, is more pronounced by 30 days of age, and is maintained after puberty at 50 days of age. In tissue from female rats, hGH does not depress the CLN-induced surge in GH release at any of the ages examined. Thus, in female rats, GH feedback does not develop, and the ability to respond to the α₂-adrenergic stimulation with a GH surge declines after puberty. Because there is no difference between male and female sensitivity to perifused GRF after 10 days of age, it is suggested that GRF insensitivity is not the mechanism for the gender differences in the CLN response. The results of this study suggest that the sexually dimorphic differences in the regulation of GH release reported here may contribute to differences between the male and female pattern of GH release after puberty.

Restenosis limits long-term success of coronary angioplasty (PTCA). Intracoronary brachytherapy, while thought to provide a solution, has also been associated with late thrombotic occlusion. We hypothesized that platelets, with their associated hemostatic, immune modulatory, and inflammatory properties, are associated with these adverse reactions. Whole blood samples were collected from consented patients undergoing de novo PTCA with stent placement (n=57), or PTCA for stent restenosis followed by intracoronary β-radiation (n=18) or γ-radiation (n=22) at baseline (BL) and at 12-24 hrs, 4–6 wks, 6 months and 1 yr post-procedure. All patients were treated with heparin, a GPIIb/IIIa inhibitor, clopidogrel and aspirin prior to PTCA. Inflammatory activation was assessed in terms of plasma IL-6 and C-Reactive Protein (CRP) levels. Platelet activation was assessed in terms of platelet P-selectin expression and the formation of platelet-monocyte complexes using flow cytometry. A significant increase in the levels of platelet-monocyte complexes (β>de novo PTCA>γ) and IL-6 (β=γ>de novo stent) 12–24 hours post-PTCA has previously been shown while levels of P-selectin (+) platelets and CRP were not significantly different between the 3 groups at any time point [Circ. 106(19):II-621, 2002]. This patient population is now further analyzed in relation to the occurrence of restenosis during the 1-year post-procedure follow-up period. Clinical follow-up was available on 76 of 97 patients (78%). Thirty-five of 76 patients exhibited some degree of late restenosis as detected by angiogram. The groups of patients with or without restenosis during the 1-year follow-up were evenly matched in terms of the incidence of diabetes (50% vs. 49%), hypertension (94% vs. 95%) and hyperlipidemia (85% vs. 90%). Patients experiencing restenosis during the follow-up period exhibited similar IL-6 levels as those patients who did not have restenosis. Although CRP levels were higher at BL and 12–24 hrs post-procedure in patients experiencing late restenosis (9.7±2.2 and 12.7±2.9 mg/ml, respectively) compared to patients without late restenosis (6.1±1.0 and 9.1±1.4 mg/ml, respectively), this difference did not reach statistical significance. Levels of platelet-monocyte complexes were increased relative to BL at 12–24 hours post-procedure (18.5±3.3% vs. 39.5±4.2%, p<0.05 vs. BL) in patients with restenosis at follow-up as well as in those without restenosis (15.5±2.4% vs. 39.4±3.5%, p<0.05 vs. BL). Platelet activation, measured as the percentage of P-selectin (+) platelets, was higher in patients experiencing restenosis at follow-up (p<0.05 vs. no restenosis). At baseline and at 12-24 hours post-procedure, the percentage of activated platelets was approximately 2-fold higher in patients who would subsequently experience restenosis (BL: 1.7±0.4 vs. 0.7±0.1%; 12–24 hrs: 2.5±0.8 vs. 1.3±0.3%). While observed with all patients, this finding was more pronounced in de novo stent patients (BL: 2.1±0.1 vs. 0.8±0.2%; 12–24 hrs: 3.4±1.5 vs. 0.9±0.3%) compared to those receiving brachytherapy. Despite potent, multi-targeted anti-platelet therapy, significant post-procedural platelet activation was observed in patients undergoing PTCA with or without subsequent brachytherapy. The data suggest that enhanced platelet activation may contribute to the restenotic process. Whether the increased platelet activation observed in the restenotic patients is due to antiplatelet drug resistance remains to be determined.