Olcese James
Institute for Hormone and Fertility Research, University of Hamburg, Hamburg, Germany.
Chronobiol Int. 2003 Jul;20(4):617-36. doi: 10.1081/cbi-120022409.
The chick pineal organ is recognized to contain an endogenous circadian oscillator as well as having direct photic input pathways and the capability of synthesizing melatonin. Despite its interesting circadian cell biology, far less is known about the chick pineal as compared to mammalian pineal glands. The goals of our research were to identify and characterize novel components of the circadian system in this photoneuroendocrine organ. Using a subtractive screening strategy of a nocturnal chick pineal cDNA library, we identified numerous genes whose expression in the chick pineal has never been reported. Among these, we focused our attention on a homologue to the regulatory subunit of the mammalian serine/threonine protein phosphatase (STPP) 2A. The expression of this gene in the chick pineal is highly circadian both in vivo and in vitro. Analysis of the PP2A enzyme in this tissue revealed that it is predominantly cytosolic in localization, sensitive to classical PP2A inhibitors, and far more active during the subjective night. Interestingly, the acute pharmacological inhibition of PP2A leads to elevated phosphoCREB levels and concomitant melatonin secretion, indicating that this enzyme participates at some level in the control of nocturnal pineal melatonin synthesis. In a second aspect of our research, we examined the mechanisms underlying the circadian rhythmicity of cyclic GMP in the chick pineal. This signaling molecule is poorly understood, despite its well-known, high-amplitude circadian rhythms and the presence of many cGMP-dependent targets in this tissue. Our work has shown that although both soluble (sGC) and membrane-bound (mGC) forms of guanylyl cyclase are present, the primary contributor to the circadian rhythms of cGMP is the mGC-B enzyme, which is activated only by the natriuretic peptide CNP. As pharmacological blockade of mGC-B (but not sGC) suppresses nocturnal cGMP levels, we conclude that CNP-dependent mechanisms are involved. Hence, the circadian clock in the chick pineal appears to drive either CNP secretion or mGC-B expression (or synthetic efficiency) in order to elevate nocturnal cGMP. Conversely, light may inhibit cGMP by uncoupling this drive. These data provide new strategies for understanding both photic input pathways (presumed to depend on cGMP) and cGMP-dependent cellular function in the chick pineal organ.
鸡的松果体器官被认为含有内源性昼夜节律振荡器，具有直接的光输入途径以及合成褪黑素的能力。尽管其昼夜节律细胞生物学很有趣，但与哺乳动物松果体相比，人们对鸡松果体的了解要少得多。我们研究的目标是鉴定和表征这个光神经内分泌器官中昼夜节律系统的新成分。通过对夜间鸡松果体cDNA文库进行消减筛选策略，我们鉴定出许多在鸡松果体中从未报道过其表达的基因。其中，我们将注意力集中在与哺乳动物丝氨酸/苏氨酸蛋白磷酸酶（STPP）2A调节亚基同源的基因上。该基因在鸡松果体中的表达在体内和体外都具有高度的昼夜节律性。对该组织中PP2A酶的分析表明，它主要定位于细胞质中，对经典的PP2A抑制剂敏感，并且在主观夜间活性更高。有趣的是，PP2A的急性药理学抑制导致磷酸化CREB水平升高和褪黑素分泌增加，这表明该酶在某种程度上参与了夜间松果体褪黑素合成的控制。在我们研究的第二个方面，我们研究了鸡松果体中环状GMP昼夜节律的潜在机制。尽管这种信号分子具有众所周知的高振幅昼夜节律以及该组织中存在许多依赖cGMP的靶点，但其作用机制仍知之甚少。我们的工作表明，虽然可溶性（sGC）和膜结合（mGC）形式的鸟苷酸环化酶都存在，但cGMP昼夜节律的主要贡献者是mGC-B酶，它仅由利钠肽CNP激活。由于对mGC-B（而非sGC）的药理学阻断会抑制夜间cGMP水平，我们得出结论，涉及依赖CNP的机制。因此，鸡松果体中的昼夜节律时钟似乎驱动CNP分泌或mGC-B表达（或合成效率）以提高夜间cGMP水平。相反，光可能通过解除这种驱动来抑制cGMP。这些数据为理解鸡松果体器官中的光输入途径（推测依赖于cGMP）和依赖cGMP的细胞功能提供了新的策略。
