Troglitazone improves blood flow by inhibiting neointimal formation after balloon injury in Otsuka Long-Evans Tokushima fatty rats.

Troglitazone (TGZ) is an antidiabetic agent of the thiazolidinedione (TZD) class that potentiates insulin action. In addition to its effects on insulin action, TGZ has an antiproliferative effect on vascular smooth muscle cells (VSMCs), of which proliferation is a prominent feature of retenosis after balloon injury, as well as atherosclerosis. Therefore, we investigated the effects of TGZ on intimal formation and blood flow after balloon injury in insulin-resistant Otsuka Long-Evans Tokushima Fatty (OLETF) rats to see whether the decrease in insulin resistance could minimize VSMC proliferation and could maintain blood flow. OLETF rats, an animal model of type 2 diabetes, develop spontaneous hyperglycemia after the age of 24 weeks. Balloon injury was applied to the left common carotid arteries of the rats with a 2F Fogarty catheter. Two weeks after the balloon injury, blood flow velocity was measured with Doppler ultrasonography, and histomorphometric analyses of the common carotid arteries were performed. The neointimal formation caused by VSMC proliferation was inhibited by TGZ treatment by as much as 80% (0.197 +/- 0.013 mm(2) v 0.157 +/- 0.011 mm(2), P <.05). The ratio of neointimal to medial area also decreased by 22% with TGZ treatment (1.651 +/- 0.148 v 1.292 +/- 0.083, P <.05). These effects of TGZ in OLETF rats were accompanied by alterations in plasma insulin, triglyceride, and total cholesterol levels. To look into the relationship between VSMC proliferation and hyperinsulinemia, we used a [(3)H]-thymidine incorporation assay to investigate the effects of TGZ on VSMC proliferation. Insulin (at a concentration of 17.3 nmol/L) significantly stimulated DNA synthesis (236.6% +/- 7.4%, P <.001), and TGZ significantly inhibited the insulin-induced DNA synthesis in VSMCs (106.43% +/- 4.23%, P <.001) in a dose-dependent manner. In balloon-injured arteries of the untreated group, systolic blood flow velocity decreased by 61% compared with uninjured arteries (P <.05). However, there was no significant difference in systolic blood flow velocity between injured and uninjured arteries in the treated group (0.906 +/- 0.043 v 0.991 +/- 0.066 meters per second [m/s], P = not significant [NS]). The systolic blood flow of injured arteries was improved by 143% in the treated group (P <.01). These data suggest that TGZ is a potent inhibitor of VSMC proliferation both in vivo and in vitro through a direct effect on VSMCs, and that TZDs might be very useful in the treatment and prevention of restenosis after balloon injury.

M3 subtype of muscarinic receptors mediate Ca2+ release from intracellular stores in rat prostate neuroendocrine cells.

Our previous studies document the expression of adrenoceptors and purinoceptors in the rat prostate neuroendocrine cells (RPNECs). However, a direct investigation of the receptors for acetylcholine (ACh) is still lacking in the prostate neuroendocrine cells. RPNECs were freshly isolated from the ventral lobes of rat prostate by using collagenase. Effects of ACh and various muscarinic antagonists on the intracellular Ca(2+) concentration ([Ca(2+)](c)) were investigated by using the fura-2 spectrofluorimetry. Single-cell RT-PCR analysis was applied to identify the transcripts for the muscarinic receptor subtypes. ACh (5 microM) induced a sharp transient increase in the [Ca(2+)](c) of RPNECs, which was independent of the extracellular Ca(2+). In the same RPNECs, high KCl (60 mM), phenylephrine (5 microM), UTP (P2Y(1/2) agonist, 50 microM), and alpha, beta-meATP (P2X(1/3) agonist, 0.5 microM) also increased the [Ca(2+)](c). The ACh-induced [Ca(2+)](c) change (delta [Ca(2+)](c)) was blocked by atropine or by para-fluorohexahydrosiladifenidol (M(3) antagonist, 0.3 microM), but not by telenzepine (M(1) antagonist, 1 microM) and himbacine (M(2) and M(4) antagonist, 1 microM). The single-cell RT-PCR demonstrated the selective expression of mRNAs for M(3) in RPNECs. In summary, RPNECs express M(3) muscarinic receptors that are linked to the release of Ca(2+) from intracellular stores. The Ca(2+) signals of RPNECs might mediate the parasympathetic regulation of prostate gland.

Purinergic receptors coupled to intracellular Ca2+ signals and exocytosis in rat prostate neuroendocrine cells.

Rat prostate neuroendocrine cells (RPNECs) display a variety of ion channels and exhibit alpha-adrenergic regulation of cytosolic Ca(2+) concentration ([Ca(2+)])(c). In this study, purinergic regulation of [Ca(2+)](c) and exocytosis was investigated in freshly isolated single RPNECs showing chromogranin A immunoreactivity. The presence of P2X and P2Y receptors in RPNECs was verified by the transient activation of Ca(2+)-permeable cationic channels and the release of Ca(2+) from intracellular stores by extracellular ATP, respectively. The transient inward cationic current was effectively activated by alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP) and blocked by 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, suggesting the presence of a P2X(1) or P2X(3) subtype. For the release of stored Ca(2+), ATP and UTP were equally potent, indicating the functional expression of the P2Y(2) or P2Y(4) subtype. The mRNAs for P2X(1) and P2Y(2) were confirmed from reverse transcription-PCR analysis of RPNECs. The application of alpha,beta-MeATP induced large and transient increases in [Ca(2+)](c), which were not attenuated by the blockers of voltage-activated Ca(2+) channels or by depleting intracellular Ca(2+) stores, but were abolished by omitting extracellular Ca(2+). The application of UTP increased [Ca(2+)](c) to 55% of the peak Delta[Ca(2+)](c) induced by alpha,beta-MeATP. The application of alpha,beta-MeATP induced exocytotic responses of RPNECs as monitored by carbon fiber amperometry and capacitance measurements. To our interest, the application of UTP did not induce amperometric currents, but reduced the membrane capacitance, indicating a net endocytosis. From these results, we postulate that a sharp rise in [Ca(2+)](c) by the P2X-mediated Ca(2+) influx is required for exocytosis, whereas the relatively slow release of stored Ca(2+) induces endocytosis in RPNECs.