Localization of cGMP-dependent protein kinase type II in rat brain.

In brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMP-dependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization.We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP effects in brain requiring protein phosphorylation.     

Cytoplasmic localization of mPER1 clock protein isoforms in the mouse retina

The mammalian Period1 gene is rhythmically expressed and its proteins are found within the nucleus of the cells of the suprachiasmatic nuclei (SCN), the central circadian pacemaker in mammals; however, whether the target of the PER1 proteins is also the nucleus in the retinal peripheral clock cells is yet to be determined. Using an anti-PER1 protein antibody in Western blot analyses, we found three isoforms (75, 110 and 140 kDa) in extracts of the SCN, as well as in other different parts of the brain, whereas just two isoforms (75 and 110 kDa) were detected in the retinal extracts. We have observed that PER1 immunolabelling has a cytoplasmic location in many cells of the ganglion cell layer and in a few cells in the inner nuclear layer of the mouse retina. This cellular location was seen in any of the tissue samples taken at 4 h intervals, either in the day/night cycle or in constant darkness, of both wild type and rd mice. Unlike this situation, PER1 isoforms were nuclear proteins in the SCN cells as well as in other parts of the brain of the same animals. No circadian changes were found for these clock proteins in the neural retina. These findings suggest that PER1 proteins play roles in the retina different from those established in the SCN.     

Spatiotemporal expression of four isoforms of Ca2+/calmodulin-dependent protein kinase I in brain and its possible roles in hippocampal dendritic growth

Among multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs), CaMKI has been shown to comprise a family of four structurally related isoforms (alpha, beta, gamma, and delta) encoded by separate genes with abundant expression in mature brain. In this study, we first examined the developmental gene expression of the four isoforms of CaMKI in mouse brain with special attention to the hippocampal formation by in situ hybridization analysis. The four isoforms of CaMKI were found to exhibit distinct spatiotemporal expression during neuronal development. We also examined the functional involvement of CaMKI in the dendritic formation of cultured hippocampal neurons. The overexpression of kinase-dead mutants of CaMKI reduced the average dendritic length of the transfected neurons without any significant effects on the number of primary dendrites and the branching index. Our present findings provide the detailed anatomical information on the developmental expression of the four isoforms of CaMKI in mouse brain, which represents the possible functional involvement of CaMKI in the basal dendritic growth of hippocampal neurons.     

Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase

Trisubstituted pyrrole inhibitors of the essential coccidian parasite cGMP dependent protein kinase (PKG) block parasite invasion and show in vivo efficacy against Eimeria in chickens and Toxoplasma in mice. An imidazopyridine inhibitor of PKG activity with greater potency in both parasite invasion assays and in vivo activity has recently been identified. Susceptibility experiments with a Toxoplasma knock-out strain expressing a complementing compound-refractory PKG allele ('T761Q-KO'), suggest a role for additional secondary protein kinase targets. Using extracts from this engineered T. gondii strain and a radiolabeled imidazopyridine ligand, a single peak of binding activity associated with calmodulin-like domain protein kinase (CDPK1) has been identified. Like PKG, CDPK1 has been implicated in host cell invasion and exhibits sub-nanomolar sensitivity to the compound. Amino acid sequence comparisons of coccidian CDPKs and a mutational analysis reveal that the binding of the ligand to PKG and CDPK1 (but not other CDPK isoforms) is mediated by similar contacts in a catalytic site hydrophobic binding pocket, and can be blocked by analogous amino acid substitutions. Transgenic strains over-expressing a biochemically active but compound-refractory CDPK1 mutant ('G128Q') fail to show reduced susceptibility to the compound in vivo, suggesting that selective inhibition of this enzyme is not responsible for the enhanced anti-parasitic potency of the imidazopyridine analog. An alternative secondary target candidate, the alpha-isoform of casein kinase 1 (CK1alpha), shows sensitivity to the compound in the low nanomolar range. These results provide an example of the utility of the Toxoplasma model system for investigating the mechanism of action of novel anticoccidial agents.     

Immunoreactive GnRH type I receptors in the mouse and sheep brain

Gonadotropin Releasing Hormone-I (GnRH) has been implicated in an array of functions outside the neuroendocrine reproductive axis. Previous investigations have reported extensive GnRH binding in numerous sites and this has been supported by in situ hybridization studies reporting GnRH receptor mRNA distribution. The present study on mice and sheep supports and extends these earlier investigations by revealing the distribution of cells immunoreactive for the GnRH receptor. In addition to sites previously shown to express GnRH receptors such as the hippocampus, amygdala and the arcuate nucleus, the improved resolution afforded by immunocytochemistry detected cells in the mitral cell lay of the olfactory bulb as well as the central grey of the mesencephalon. In addition, GnRH receptor immunoreactive neurons in the hippocampus and mesencephalon of the sheep were shown to colocalize with estrogen receptor beta. Although GnRH may act at some of these sites to regulate reproductive processes, evidence is accumulating to support an extra-reproductive role for this hypothalamic decapeptide.     

Calcium/calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain

A second isoform of Ca2+/calmodulin-dependent-kinase II inhibitor protein (CaM-KIIN) has been identified using the yeast two-hybrid screen. The 1.8kb message encodes a 78 residue CaM-KIINalpha that is 65% identical in its putative open-reading frame and 95% identical in its inhibitory domain to the previously characterized CaM-KIINbeta. CaM-KIINalpha exhibits inhibitory properties towards recombinant mouse CaM-kinase IIalpha indistinguishable from CaM-KIINbeta. The 27 amino acid inhibitory peptide (CaM-KIINtide) derived from CaM-KIIN has the ability to inhibit brain CaM-kinase II activity from multiple organisms including rat, Drosophila and goldfish. Northern analysis of various rat tissues indicates that CaM-KIINalpha is specific to brain whereas CaM-KIINbeta message is also present in testis. In situ hybridization shows a general distribution of both isoforms in rat brain with stronger localization of CaM-KIINbeta in cerebellum and hindbrain and CaM-KIINalpha in frontal cortex, hippocampus and inferior colliculus. An antibody that recognizes both isoforms shows a distribution of CaM-KIIN in rat brain that correlates with immunoreactivity of CaM-kinase II. In cultured mature hippocampal neurons, CaM-KIIN is present in cell bodies and dendrites but, unlike CaM-kinase II, does not display punctate staining at synapses. These results suggest a localized function for CaM-KIIN in inhibiting specialized pools of CaM-kinase II.     

Function of cGMP-dependent protein kinase II in volume load-induced diuresis

Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3 % of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50 % in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII.     

Localization of Triton-insoluble cAMP-dependent kinase type RIbeta in rat and mouse brain

In eukaryotic cells, cAMP regulates many different cellular functions. Its effects are in most cases mediated by cAMP-dependent protein kinases. These consist of two regulatory and two catalytic subunits. In mammals, four different isoforms of cAMP-dependent protein kinases regulatory subunits have been characterized (RI and , RII and ). These four isoforms show a high level of homology and slightly different biochemical properties. In addition to biochemical properties, a different anatomical distribution of the regulatory isoforms may contribute to determine the specificity of diverse cAMP effects. By immunohistochemistry, the distribution of the detergent-insoluble fraction of RI isoform has been examined in rat and mouse brain. Biochemical fractionation shows that a large fraction of both RI and RI isoforms is bound to the cytoskeleton. RI labelling can be observed only in few locations: Purkinje cells, olfactory mitral cells, lateral thalamic neurons, superior olivary complex neurons. These cell populations are involved in the so called Purkinje cell degeneration. On the other hand, RI aggregates have a more widespread distribution, in brain areas involved in visceroemotional control. At the subcellular level, these two subunits show a different pattern of labelling: in most cells a sharply defined clustered labelling is observed for RI isoforms, while the RI isoform presents a weaker, diffuse intracytoplasmic distribution. Competition experiments point to the presence of, as yet unidentified, different and selective anchoring proteins for the two similar RI and isoforms. It is suggested that, as is the case for structural proteins, a different supramolecular organization of similar regulatory proteins may be crucial in order to fulfill different functions.     

蛋白激酶G介导烧伤休克后内皮细胞骨架的变化

目的:探讨蛋白激酶G(PKG)在烧伤休克发病机制中的作用。方法:用10%人烧伤血清刺激培养的内皮细胞后,通过细胞裂解和离心获得细胞裂解液,用放射性同位素法测定PKG的活性。同时采用特异性荧光染色法检测细胞内肌动蛋白微丝(F-actin)的结构和分布变化。用PKG特异性抑制剂KT5823预处理细胞后,再检测烧伤血清介导的细胞内PKG活性和F-actin的变化。以空白组为阴性对照,以PKG激动剂8-Br-cGMP刺激细胞作为阳性对照组。结果:人烧伤血清和PKG的激动剂8-Br-cGMP都可以时间依赖性地激活内皮细胞的PKG,并且诱导细胞内F-actin呈极性分布。KT5823预处理则明显抑制了这种变化。结论:烧伤血清可以介导血管内皮细胞PKG的激活和F-actin的应力性变化;烧伤休克时的血管通透性升高与PKG的活性变化密切正相关,抑制PKG活性有可能防治大面积烧伤后的血管通透性升高。     

Immunocytochemical staining with antibodies against protein kinase C and its isozymes in the turtle retina

Summary An LM immunocytochemical study has investigated the patterns of staining in turtle retina with monoclonal antibodies to the , and isozymes of protein kinase C. The protein kinase C- antibody reveals cells in the ganglion cell layer, occasional amacrine cells and faint banding in strata 2 and 4 of the inner plexiform layer. The protein kinase C- antibody stains primarily amacrine cells that have dendrites running in strata 2, in 4 close to the 3/4 border and on the 4/5 border of the inner plexiform layer. Protein kinase C- immunoreactivity is seen in a population of bipolar cells. The latter are characterized by stained axon terminals in strata 3 and 4 of the inner plexiform layer. A type of amacrine cell, different from those seen with the other antibodies, is also immunoreactive to protein kinase C-. EM immunocytochemistry (using a polyclonal antibody) reveals protein kinase C immunoreactivity in photoreceptor cells, bipolar cells, amacrine cells and ganglion cells. In photoreceptors protein kinase C immunoreactivity occurs as patchy staining associated with vesicles and the plasmalemma in pedicles and telodendria. Some varieties of bipolar cell display protein kinase C reaction product throughout the entire cell. Their dendrites contact photoreceptor pedicles at wide-cleft basal junctions and ribbon and non-ribbon related narrow cleft junctions. A few lateral elements per cone or rod pedicle are always protein kinase C-immunoreactive. Amacrine and ganglion cells typically show small clumps of protein kinase C immunoreactivity around vesicles and close to the postsynaptic membranes. Synaptic boutons of some varieties of amacrine cell stain more uniformly. Protein kinase C-immunoreactive bipolar cells are most commonly presynaptic in stratum 4 of the inner plexiform layer, while protein kinase C-immunoreactive amacrine cells are both pre- and postsynaptic throughout strata 1, 2, 3 and 4. Stratum 5 appears to be almost devoid of protein kinase C-immunoreactive neural profiles.     

Dual regulation of the ATP-sensitive potassium channel by activation of cGMP-dependent protein kinase

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels couple cellular metabolic status to membrane electrical activity. In this study, we performed patch-clamp recordings to investigate how cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) regulates the function of K(ATP) channels, using both transfected human SH-SY5Y neuroblastoma cells and embryonic kidney (HEK) 293 cells. In intact SH-SY5Y cells, the single-channel currents of Kir6.2/sulfonylurea receptor (SUR) 1 channels, a neuronal-type K(ATP) isoform, were enhanced by zaprinast, a cGMP-specific phosphodiesterase inhibitor; this enhancement was abolished by inhibition of PKG, suggesting a stimulatory role of cGMP/PKG signaling in regulating the function of neuronal K(ATP) channels. Similar effects of cGMP accumulation were confirmed in intact HEK293 cells expressing Kir6.2/SUR1 channels. In contrast, direct application of purified PKG suppressed rather than activated Kir6.2/SUR1 channels in excised, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit were not modulated by zaprinast or purified PKG. Lastly, reconstitution of the soluble guanylyl cyclase/cGMP/PKG signaling pathway by generation of nitric oxide led to Kir6.2/SUR1 channel activation in both cell types. Taken together, here, we report novel findings that PKG exerts dual functional regulation of neuronal K(ATP) channels in a SUR subunit-dependent manner, which may provide new means of therapeutic intervention for manipulating neuronal excitability and/or survival.     

Plasmin-mediated processing of protein tyrosine phosphatase receptor type Z in the mouse brain

Protein tyrosine phosphatase receptor type Z (Ptprz, also known as PTPzeta or RPTPbeta) is preferentially expressed in the CNS as a major chondroitin sulfate proteoglycan (CSPG). Ptprz interacts with the PSD95 family through its intracellular carboxyl-terminal PDZ-binding motif in the postsynaptic density. Ptprz-deficient adult mice display impairments in spatial and contextual learning. Here, we identified the proteolytic processing of Ptprz by plasmin in the mouse brain, which is markedly enhanced after kainic acid (KA)-induced seizures. We mapped plasmin cleavage sites in the extracellular region of Ptprz by cell-based assays and in vitro digestion experiments with recombinant proteins. These findings indicate that Ptprz is a physiological target for activity-dependent proteolytic processing by the tPA/plasmin system, and suggest that the proteolytic cleavage is involved in the functional processes of the synapses during learning and memory.     

Localization of the cGMP-dependent protein kinases in relation to nitric oxide synthase in the brain

The distributions of the type I and type II isoforms of cGMP-dependent protein kinase were determined in the rat brain using immunohistochemistry and in situ hybridization, and compared with the localization of NO synthase determined with NADPH-diaphorase histochemistry. The type I cGMP-dependent protein kinase was highly expressed in the Purkinje cells of the cerebellar cortex, where it was closely associated with the NO synthase containing granule and basket cells. This kinase was also found in neurons in the dorsomedial nucleus of the hypothalamus, where it may be regulated by NO or atriopeptides. The type I kinase was not detected in other central neurons. In contrast, the type II kinase was widely distributed in the brain. In particular, it was highly expressed in the olfactory bulb, cortex, septum, thalamus, tectum and various brainstem nuclei. Many regions expressing this kinase also contained, or received innervation from NO synthase positive neurons. These results indicate that type I cGMP-dependent protein kinase may act as a downstream effector for NO only in the cerebellar cortex and the dorsomedial hypothalamus. The type II cGMP-dependent protein kinase appears to be a major mediator of NO actions in the brain.     

Inhibition of cGMP-dependent protein kinase reduces the response to sonic hedgehog in neuralized embryoid bodies

Previously, we have shown that increasing the intracellular cGMP concentration enhances the sonic hedgehog (Shh) response in neural plate cells. The use of two mouse embryonic stem (ES) cell lines allowed a highly sensitive and reproducible quantification of the Shh response in neuralized embryoid bodies. Here we demonstrate that the specific, membrane-permeable cGMP-dependent protein kinase G-Ialpha (PKG-Ialpha) inhibitor DT-2 prevents an efficient Shh response, indicating that the effects of cGMP on the Shh response are mediated via PKG. We also demonstrate that the PKG acts upon the Shh response upstream of the Ptc1 promoter, which is up-regulated invariably and early in response to Shh, significantly limiting the targets for PKG phosphorylation to molecules involved in the early steps of the Shh response. These effects of cGMP and PKG are antagonistic to those of cAMP and PKA, and thus provide a mechanism by which the sensitivity of cells to the effects of Shh can be regulated, by modulating the intracellular cyclic nucleotide concentration.     

Immunohistochemical localization of protein kinase C (PKC) beta I in the pig retina during postnatal development

In order to investigate the expression of protein kinase C (PKC) beta I in the retinas of pigs during postnatal development, we analyzed retinas sampled from 3-day-old and 6-month-old pigs by Western blotting and immunohistochemistry. Western blot analysis detected the expression of PKC beta I in the retinas of 3-day-old piglets and it was increased significantly in the retinas of 6-month-old adult pigs. Immunohistochemical staining showed PKC beta I in the retinas of both groups. Immunohistochemistry of 3-day-old retinas revealed weak PKC beta I reactivity in the ganglion cell layer, inner plexiform layer, inner nuclear cell layer, outer plexiform layer and rod and cone cell layer. In the 6-month-old pig retina, the cellular localization of PKC beta I immunostaining was similar to that of the 3-day-old retina, where PKC beta I was localized in some glial fibrillary acidic protein-positive cells, glutamine synthetase-positive cells, parvalbumin-positive cells, and PKC alpha-positive cells in the retina. This is the first study to show the expression and cellular localization of PKC beta I in the retina of pigs with development, and these results suggest that PKC beta I, in accordance with PKC alpha, plays important roles in signal transduction pathways in the pig retina with development.     

cGMP facilitates calcium current via cGMP-dependent protein kinase in isolated rabbit ventricular myocytes

 The effect of guanosine 3′,5′-cyclic monophosphate (cGMP) on L-type Ca current (I _Ca) was investigated in a study of rabbit ventricular myocytes using the whole-cell patch-clamp technique. Intracellular application of cGMP (100 μM) increased I _Ca in the absence of isoprenaline or forskolin. 8-Bromo-cGMP (100 μM) and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP, 400 μM), relatively specific stimulators of cGMP-dependent protein kinase (cGMP-PK), also increased I _Ca. The stimulatory effect of 8-pCPT-cGMP was suppressed by Rp-8-chlorophenylthio-cGMP (400 μM), a phosphodiesterase-resistant cGMP-PK inhibitor. When I _Ca was increased by bath application of the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM), 8-pCPT-cGMP (400 μM) resulted in additional stimulation of I _Ca. In the presence of 8-pCPT-cGMP, additional applications of isoprenaline (1 μM) or forskolin (1 μM) induced a further increase in I _Ca. From these results, it could be concluded that the activation of cGMP-dependent protein kinase is involved in the facilitation of I _Ca by cGMP in rabbit ventricular myocytes. Received: 17 March 1997 / Received after revision: 28 August 1997 / Accepted: 16 September 1997