Cohen D P, Rothstein T L
Department of Microbiology, Boston University Medical Center, MA 02118.
J Immunol. 1991 May 1;146(9):2921-7.
Conventional murine splenic B cells are stimulated to initiate DNA synthesis by the combination of a phorbol ester protein kinase C (PKC) agonist, and a calcium ionophore; in contrast, recent work from this laboratory has shown that peritoneal B cells, enriched for the Ly-1+ B cell subset, differ in that they proliferate in response to the single signal provided by phorbol ester, acting alone. To elucidate the mechanism responsible for the abbreviated signaling requirement of peritoneal B cells, studies of intracellular Ca2+ and PKC were carried out. Measurements using the calcium sensitive dye, Indo-1, showed that base line levels of intracellular Ca2+ in peritoneal B cells were similar to those of splenic B cells, and that there was no change as a result of phorbol ester treatment. However, measurements of PKC based on the phosphorylation of histone showed enzymatic activity in peritoneal B cells to be about 60% greater than that of splenic B cells on a per microgram protein basis. Furthermore, this difference was accentuated by phorbol ester treatment, so that after 4 h, membrane and cytosol fractions from peritoneal B cells contained more than 5 times the PKC activity of the corresponding splenic B cell fractions because the down-regulation of PKC was relatively delayed in peritoneal B cells. This could not be accounted for by the onset of new PKC synthesis, but may relate to the finding that peritoneal B cells express more of the alpha-isoenzyme of PKC than splenic B cells, as shown by immunoblot analysis. Together with data from experiments using the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride(H7), these results suggest that PKC activity remaining hours after phorbol ester treatment may contribute to the unusual phorbol ester responsiveness of peritoneal B cells, and indicate that B cells from separate anatomic locations differ in terms of several parameters relating to the activity and behavior of PKC.
传统的小鼠脾B细胞通过佛波酯蛋白激酶C（PKC）激动剂和钙离子载体的联合作用被刺激启动DNA合成；相比之下，本实验室最近的研究表明，富含Ly-1+B细胞亚群的腹膜B细胞有所不同，它们仅对佛波酯单独提供的单一信号作出增殖反应。为了阐明腹膜B细胞信号传导需求简化的机制，我们对细胞内Ca2+和PKC进行了研究。使用钙敏感染料Indo-1进行的测量表明，腹膜B细胞内Ca2+的基线水平与脾B细胞相似，并且佛波酯处理后没有变化。然而，基于组蛋白磷酸化的PKC测量表明，以每微克蛋白质计算，腹膜B细胞中的酶活性比脾B细胞高约60%。此外，佛波酯处理加剧了这种差异，以至于4小时后，腹膜B细胞的膜和胞质部分所含的PKC活性是相应脾B细胞部分的5倍多，因为腹膜B细胞中PKC的下调相对延迟。这不能用新PKC合成的开始来解释，但可能与免疫印迹分析所示的腹膜B细胞比脾B细胞表达更多PKCα同工酶这一发现有关。与使用PKC抑制剂1-（5-异喹啉磺酰基）-2-甲基哌嗪二盐酸盐（H7）的实验数据一起，这些结果表明佛波酯处理数小时后仍存在的PKC活性可能导致腹膜B细胞对佛波酯的异常反应，并表明来自不同解剖位置的B细胞在与PKC活性和行为相关的几个参数方面存在差异。
