Abstract. Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (S, e, e', and~) as well as the calcium-dependent isoforms (a, 01, ßn, and -y).These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised . Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide . PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography . Peak I reacted exclusively with antisera to PKCy, peak II with PKCß, and -ßu, and peak III with PKCa. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinc-P ROTEIN kinase C (PKC)' plays a major role in transmembrane signal transduction (35). PKC is activated by diacylglycerol, which is generated from membrane phospholipids upon stimulation of cells with various agonists (4) . PKC also serves as the receptor for phorbol esters and related tumor promoters (34), which activate the kinase by substituting for endogenous diacylglycerol (8) . Because of the pleiotropic actions of diacylglycerol and phorbol esters, PKC has been implicated in the regulation ofa variety of cellular processes, including proliferation, differentiation, and release of hormones and neurotransmitters (35,36).Molecular cloning studies have revealed that PKC consists of a large family of at least eight different isoenzymes (36, 37) that can be divided into two major groups. Initially, four isoforms of PKC were described . This group consists of PKCca, -01, -ß, 1, and -y, with PKCß, and -ß arising via alternate splicing of the same gene transcript and resulting in distinct carboxy-terminal regions . All four of these isoen-1. Abbreviations used in this paper: PKC, protein kinase C.O The Rockefeller University Press,

The key roles of the excitatory neurotransmitter glutamate and its second messengers, nitric oxide (NO) and cGMP, in long‐term potentiation and neural plasticity are well documented. However, complex functions such as memory are likely to require long term changes in synaptic efficacy which require gene expression and protein synthesis. Here we demonstrate that the glutamate receptor agonist, N‐methyl‐D‐aspartic acid (NMDA), nitric oxide (NO) and cGMP each repress expression of the gonadotropin‐releasing hormone (GnRH) gene in the hypothalamic cell line, GT1. This repression is dependent upon signals from NMDA receptors activating NO synthase to synthesize NO. In turn NO induces guanylyl cyclase to synthesize cGMP, activating cGMP‐ dependent protein kinase. Repression requires elevation of calcium because it only occurs in the presence of calcium ionophore or with release of intracellular calcium. Repression also requires protein synthesis. Activation of this pathway specifically represses expression of a reporter gene containing the regulatory region of the GnRH gene in transfected GT1 cells, indicating that repression occurs at the transcriptional level. Furthermore the target for transcriptional repression is a 300 bp neuron‐specific enhancer found 1.5 kb upstream of the GnRH gene which is sufficient to confer repression to a heterologous promoter. Thus the NMDA/NO/cGMP neurotransmitter signal transduction pathway controls not only synaptic function but also neuron‐specific gene expression.

As major signal transduction cascades, the protein kinase-A and -C (PKA and PKC) pathways have been implicated in the regulation of GnRH synthesis and secretion in the hypothalamus. We have investigated the roles of these pathways in the regulation of GnRH transcription, mRNA levels, propeptide processing, and secretion in GT1-7 cells, a mouse hypothalamic GnRH neuronal cell line. Forskolin, which activates adenylate cyclase to raise cAMP levels, had no effect on GnRH mRNA levels at 10 microM, but induced c-fos mRNA at 30 min. An activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM), also induced c-fos at 30 min, but produced a progressive decline in GnRH mRNA, resulting in a 70% decrease by 16 h. Coadministration of 10 nM TPA and 20 microM of a PKC inhibitor, NPC 15437 [2,6-diamino-N-([1-(1-oxotridecyl)2-piperidinyl]methyl)hexanami de], prevented c-fos induction, but did not antagonize GnRH repression. Instead, the inhibitor itself reduced GnRH mRNA levels by 56% at 16 h (with no effect on c-fos mRNA). Thus, since extended exposure to TPA can down-regulate PKC, suppression of GnRH mRNA by TPA may be due to decreased PKC activity, indicating a role for PKC in the maintenance of the GnRH gene expression (a role that is unlikely to involve c-fos). In transient transfections, the transcriptional activity from 3 kilobases of GnRH 5'-flanking sequence was repressed 2-fold by either 100 nM TPA or 20 microM NPC 15437 at 24 h, demonstrating that suppression of GnRH mRNA is at least, in part, at the level of transcription. In contrast, both TPA (100 nM) and forskolin (10 microM) stimulated secretion. Enhancement of GnRH secretion by TPA was robust and rapid (2.5 min), while the response to forskolin was relatively delayed (2 h). Over a 24-h period, unstimulated cells released primarily unprocessed prohormone, whereas forskolin and TPA stimulated the secretion of processed products. These data indicate that PKC and PKA may influence propeptide processing and/or the route of GnRH secretion. These data demonstrate that the PKA and PKC pathways regulate GnRH at the multiple levels of transcription, pro-GnRH processing, and GnRH secretion.

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