In this study we tested the hypothesis that extracellular ATP regulates the function of the pericyte-containing retinal microvessels. Pericytes, which are more numerous in the retina than in any other tissue, are abluminally located cells that may adjust capillary perfusion by contracting and relaxing. At present, knowledge of the vasoactive molecules that regulate pericyte function is limited. Here, we focused on the actions of extracellular ATP because this nucleotide is a putative glial-to-vascular signal, as well as being a substance released by activated platelets and injured cells. In microvessels freshly isolated from the adult rat retina, we monitored ionic currents via perforated-patch pipettes, measured intracellular calcium levels with the use of fura-2, and visualized microvascular contractions with the aid of time-lapse photography. We found that ATP induced depolarizing changes in the ionic currents, increased calcium levels and caused pericytes to contract. P2X7 receptors and UTP-activated receptors mediated these effects. Consistent with ATP serving as a vasoconstrictor for the pericyte-containing microvasculature of the retina, the microvascular lumen narrowed when an adjacent pericyte contracted. In addition, the sustained activation of P2X7 receptors inhibited cell-to-cell electrotonic transmission within the microvascular networks. Thus, ATP not only affects the contractility of individual pericytes, but also appears to regulate the spatial and temporal dynamics of the vasomotor response.

Trans-1-amino-3-18 F-fluorocyclobutanecarboxylic acid (anti-18 F-FACBC) is an amino acid PET tracer that has shown promise for visualizing prostate cancer. Therefore, we aimed to clarify the anti-18 F-FACBC transport mechanism in prostate cancer cells. We also studied the fate of anti-18 F-FACBC after it is transported into cells. Methods: For convenience, because of their longer half-lives, 14 C compounds were used instead of 18 F-labeled tracers. Trans-1-amino-3-fluoro-1-14 C-cyclobutanecarboxylic acid ( 14 C-FACBC) uptake was examined in human prostate cancer DU145 cells with the following substrates of amino acid transporters: a-(methylamino) isobutyric acid (a system A-specific substrate) and 2-amino-2-norbornanecarboxylic acid (a system L-specific substrate). The messenger RNA expression of amino acid transporters in human prostate cancer specimens was analyzed by complementary DNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression in DU145 cells was analyzed by qRT-PCR. We also examined the knockdown effect of the amino acid transporters system ASC transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter 2 (SNAT2) on 14 C-FACBC uptake. In addition, the possibility of 14 C-FACBC incorporation into proteins was examined. Results: 14 C-FACBC uptake by DU145 cells was markedly decreased to approximately 20% in the absence of Na 1 , compared with that in its presence, indicating that Na 1 -dependent transporters are mainly responsible for the uptake of this tracer. Moreover, 2-amino-2-norbornanecarboxylic acid inhibited the transport of 14 C-FACBC to the basal level in Na 1 -free buffer. In contrast, a-(methylamino) isobutyric acid did not inhibit 14 C-FACBC accumulation in DU145 cells. Human prostate tumor specimens and DU145 cells had similar messenger RNA expression patterns of amino acid transporter genes. Although SNAT2 and ASCT2 are 2 major amino acid transporters expressed in prostate tumor tissues and DU145 cells, ASCT2 knockdown using small interfering RNA was more effective in lowering 14 C-FACBC transport than SNAT2. Almost all intracellular 14 C-FACBC was recovered from the nonprotein fraction. Conclusion: ASCT2, which is a Na 1 -dependent amino acid transporter, and to a lesser extent Na 1 -independent transporters play a role in the uptake of 14 C-FACBC by DU145 cells. Among the Na 1 -independent transporters, system L transporters are also involved in the transport of 14 C-FACBC. Moreover, 14 C-FACBC is not incorporated into proteins in cells. These findings suggest a possible mechanism of anti-18 F-FACBC PET for prostate cancer.

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte-containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated-patch pipettes, measured pericyte calcium levels with fura-2 and visualized pericyte contractions and lumen constrictions by time-lapse photography. We found that angiotensin activates nonspecific cation (NSC) and calcium-activated chloride channels; the opening of these channels induces a depolarization that is sufficient to activate the voltage-dependent calcium channels (VDCCs) expressed in the retinal microvasculature. Associated with these changes in ion channel activity, intracellular calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments revealed that an influx of calcium through the NSC channels is an essential step linking the activation of AT 1 angiotensin receptors with pericyte contraction. Although not required in order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to enhance the contractile response of these cells. In addition to activating nonspecific cation, calcium-activated chloride and voltage-dependent calcium channels, angiotensin II also causes the functional uncoupling of pericytes from their microvascular neighbours. This inhibition of gap junction-mediated intercellular communication suggests a previously unappreciated complexity in the spatiotemporal dynamics of the microvascular response to angiotensin II.

The aim of this study was to test the hypothesis that the neurotransmitter acetylcholine regulates the function of pericyte-containing retinal microvessels. A vasoactive role for acetylcholine is suggested by the presence of muscarinic receptors on pericytes, which are abluminally positioned contractile cells that may regulate capillary perfusion. However, little is known about the response of retinal microvessels to this neurotransmitter. Here we assessed the effects of cholinergic agonists on microvessels freshly isolated from the adult rat retina. Ionic currents were monitored via perforated patch pipettes; intracellular Ca(2+) levels were quantified with the use of fura 2, and microvascular contractions were visualized with the aid of time-lapse photography. We found that activation of muscarinic receptors elevated pericyte calcium levels, increased depolarizing Ca(2+)-activated chloride currents and caused pericytes to contract in a Ca(2+)-dependent manner. Most contracting pericytes were near capillary bifurcations. Contraction of a pericyte caused the adjacent capillary lumen to constrict. Thus acetylcholine may serve as a vasoactive signal by regulating pericyte contractility and thereby capillary perfusion in the retina.

A diabetes-induced increase in the vulnerability of retinal microvessels to the lethal effect of P2X(7) receptor activation may be a previously unrecognized mechanism by which diabetic retinopathy progresses.